A comparison of somatic chromosomal instability in tissue culture regenerants from Medicago media Pers

Two cultivars (Heinrichs, Reaver) and two breeding lines (Br1, Le1) from Medicago media were cultured in a media protocol consisting of a high concentration 2,4-D induction step. Regenerants were produced from all four stocks. Representative samples from each regenerant population along with the corresponding control population were cytologically analyzed for chromosomal and pollen abnormalities. While numerical changes in chromosome numbers were found in all regenerant populations, there was considerable variation between the four stock groups. Heteroploidy was observed for both hypo and hyper aneuploid regenerants, but there were no differences in pollen stainability between hypo and hyper aneuploid regenerants and euploid regenerants. Tissue culture regenerants generally produced a lower pollen stainability percent as compared to control populations grown from seeds. Gross and cryptic changes in chromosomes, or hormonal carry over effects or both were considered causes for poor pollen stainability in tissue culture regenerants. Cytological analyses indicate that the cultivar might play an important role in the cytological stability or instability of regenerant populations. Exploitation of this naturally existing situation to produce euploid regenerants for field experiments and to obtain gross cytological stability in somaclones is discussed.     

Somaclonal variation in wheat: genetic and cytogenetic characterisation of alcohol dehydrogenase 1 mutants

Summary The progeny of 551 regenerants of the hexaploid wheat cultivar Millewa were analysed for somaclonal mutants at the threeAdh-1 loci in hexaploid wheat. Seventeen regenerants gave rise to progeny having altered ADH1 zymograms. Progeny with altered zymograms in 13 of these regenerants were aneuploid. The remaining 4 regenerants gave rise to euploid progeny with altered ADH1 zymograms. The genetics of three of these somaclonal mutants is described in detail. These regenerants were interpreted to possess a 4A isochromosome, a 3BS/4A translocation and a 7BS/4A translocation, respectively.This research was partly supported by the Rockefeller Foundation and a Reserve Bank of Australia Rural Credit Development Fund research grant     

Aneuploid and alloplasmic lines as tools for the study of nuclear and cytoplasmic control of culture ability and regeneration of scutellar calli from common wheat

Summary Twenty four B genome aneuploid lines (di-telosomics, nullisomic-tetrasomics and tetrasomics) of Triticum aestivum cv Chinese Spring were used in an analysis of the culture ability and regeneration capability of scutellar calli. Several correlations were found between the presence or absence of specific chromosomes and chromosomal arms of the B genome of common wheat and the growth and differentiation capabilities of these calli. The rate of callus growth decreased only when the long arm of chromosome 6B was not present. The absence of chromosomes 3B and 7B did not result in an apparent change in morphogenetic capability, while the absence of other B genome chromosomes was significantly correlated to changes in the frequency of calli that regenerated plants. The presence of the short arm of chromosome 1B was negatively correlated with regeneration, whereas its long arm is probably required to counteract this effect and to maintain the normal ratio of regeneration. The presence of the chromosomal arm 2BS seemed to be essential for differentiation to shoots. In the absence of the short arms of chromosomes 4B and 5B, the rate of regeneration was slightly reduced. In the absence of the long arm of chromosome 6B there was a marked reduction of the ability of scutellar calli to regenerate plants. The use of additional aneuploid lines belonging to homoeologous group 6 revealed that only calli derived from lines having chromosome 6D in their complement regenerated plants similarly to the euploid control. Culture ability and regeneration capability were also analysed with alloplasmic lines of T. aestivum cv Chris. The lines were derived from five species, representing plasma-types of different phylogenetic distances from plasma-type B of T. aestivum. The results showed that when the endogenous cytoplasm (B-type) was exchanged with T. timopheevii cytoplasm (G-type) there was a significant increase in the regeneration of shoots from the scutellar calli.     

Chromosome variation in wheat plants regenerated from cultured immature embryos

Summary A cytological study has been made of plants regenerated from cultured immature embryos of four wheat cultivars (Triticum aestivum, 2n = 6x = 42). In total, 29% of the 192 plants examined were aneuploid with a range in chromosome numbers of 38–45. Evidence of chromosome structural changes was also found. This variation occurred in regenerants of all four cultivars, but there were large differences in the proportions of aneuploids arising from individual cultures which meant that no significant differences could be demonstrated between cultivars. Chromosome abnormalities were present in plants regenerated both from embryogenic cultures and from cultures in which the origin of shoots could not be distinctly defined.     

Adventitious shoot formation from leaf explants of Rhododendron

A protocol for high frequency adventitious shoot regeneration adventitious shoot regeneration from leaf explants of Rhododendron spp. has been developed. The highest percentage of regeneration and the greatest number of shoots were obtained when leaf explants were cultured on Anderson's medium containing 4.9 M IBA and 73.8 M 2iP. Genotypic variation was observed for adventitious shoot regeneration potential among the seven cultivars tested. Regeneration frequencies ranged from 0 to 96%. Lodestar had the highest rate of regeneration after 3 months of culture with 96% shoot regeneration and an average of 14 shoots per explant. Regenerated shoots were rooted in soil in about 2 months. This protocol should be useful in applying gene transfer techniques to Rhododendron improvement.Abbreviations IAA 1-H-indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA 1-H-indole-3-butyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - 2iP N-(3-methyl-2-butenyl)-1-H-purine-6-amine     

Chromosomal localization of structural genes and regulators in wheat by 2D electrophoresis of ditelosomic lines

Summary Among the 782 spots observed in two-dimensional gel electrophoresis of denatured proteins from etiolated wheat shoots, 185 were found to be variable between the euploid and 26 ditelosomic lines of Chinese Spring. Thirty-five structural genes were located on 17 chromosome arms. Numerous intensity changes showing alterations in protein levels were observed and led to the following statements: 1) regulators are frequently found and can be assigned for a same polypeptide to various chromosome arms; 2) for most polypeptides homoeologous arms do not manifest similar effects; 3) nevertheless, when affecting the same polypeptide, homoeologous arms display in most cases identical regulatory effects; 4) gene dosage compensation is observed in only one out of four homoeoallelic situations.     

Chromosomal variation in immature embryo derived calluses of barley (Hordeum vulgare L.)

Summary Chromosome counts of ten morphogenic and seven non-morphogenic immature embryo derived calluses of barley,Hordeum vulgare L. cv. Himalaya, were determined. Morphogenic calluses carried the normal chromosome complement (2n=2x=14) in a majority of the cells. A low frequency of haploid (2n=x=7), triploid (2n=3x=21), tetraploid (2n=4x=28) and octoploid (2n=8x=56) cells were also observed. In contrast, non-regenerability of a callus was attributed to the cells having numerical and structural chromosomal changes. In these calluses, aneuploid cells around diploid, triploid, and tetraploid chromosome numbers predominated. It has been demonstrated that chromosomal changes were induced during the culture and that they did not pre-exist in the cultured barley embryos. Based on this study, it is suggested that chromosome analysis of a non-regenerable callus should be conducted before altering the media composition.     

Ploidy dependence of chromosomal variation in callus cultures ofHyoscyamus muticus L.

Summary Remarkable variation for chromosome number was observed in both diploid (2x) and autotetraploid (4x) callus cultures continuously examined for 12 months at monthly subsculture intervals. Initially, the subcultures exhibited predominantly the genomic level of the starting material which subsequently and gradually developed into heterogenous populations of euploid and aneuploid cells imparting the subcultures a aneusomatic status. The comparison of chromosomal instability recorded in 2x vs. 4x callus cultures revealed that with time both types of calli stabilized at a chromosome number around the 4x level. However, the chromosomal examination of the adventitious roots emerging from the disorganized calli revealed the euploid (both 2x and 4x) chromosome levels suggesting the occurrence of amorphogenetic sieve.This paper is dedicated to Professor A. K.Sharma of Calcutta University on his 65th birthday by his former student U. C. L. and grandstudent S. S. (CIMAP Publication no. 739).     

Factors affecting regeneration of pigeonpea (Cajanus cajan L. Millsp) from mature embryonal axes

Regeneration of viable plants was obtained via organogenesis from mature embryonal axes explants of pigeonpea. Shoots were produced from the apical region of embryonal axes after 20 days of dark incubation on modified Murashige and Skoog (1962) medium containing 8.86 M benzylaminopurine (BAP) and 1.07 M -naphthalene acetic acid (NAA). When the explants were cultured under light-dark (16--8 hrs) conditions, shoots were initiated only after 65 days of culture initiation. The explants lost their ability to regenerate plantlets when they were cultured in continuous light. Regenerated shoots elongated either in the same medium or in MS basal medium. About 40% of the elongated shoots sequentially produced simple, bifoliate and finally trifoliate leaves instead of producing trifoliate leaves directly. The elongated shoots were rooted efficiently upon transferring them to half strength MS medium supplemented with 2.41 M indole-3-butyric acid (IBA). Rooted plantlets were successfully transferred to soil after hardening.     

Cytogenetic analysis of structural rearrangements in three varieties of common wheat,Triticum aestivum

Summary The winter wheat varieties Starke and Cappelle Desprez and the spring wheat Chinese Spring were analysed for structural chromosome rearrangements that resulted in the formation of multivalents in F₁ hybrids. The analyses were carried out using hybrids involving euploids, monosomic and ditelosomic stocks, and double-monotelodisomic constructs. The study confirmed that Cappelle Desprez differs from Chinese Spring in a reciprocal translocation between chromosomes 5B and 7B (Riley et al. 1967); a translocation involving chromosomes 3B and 3D could not be verified. Furthermore, the analysis showed that Starke differs from Chinese Spring in a reciprocal translocation between chromosomes 7A and 7D. Both translocations have a coefficient of multivalent realisation of about 0.84. Further multivalents in euploid Starke, in euploid and some aneuploid stocks of Cappelle Desprez, and in euploid as well as various types of aneuploid hybrids between all three varieties could nearly all be explained hypothesizing that chromosome 2B of both Starke and Cappelle Desprez is a duplication-deficiency chromosome. In the hypothesis a part of the long arm of 2B is missing and replaced by a duplicated part of the long arm of chromosome 2D. The multivalents of this rearrangement showed an average coefficient of realisation of about 0.09.Sven Ellerström died in December 1985     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

In vitro clonal propagation of Pisum sativum L.

A system of in vitro clonal propagation has been developed in Pisum sativum L. (cv. Bohatýr). A modified MS-medium supplemented with 20 M 6-benzylaminopurine (BAP) and 0.1 M -naphthaleneacetic acid (NAA) was used to induce multiple shoot formation from shoot apices, axillary buds of the first normal leaf, axillary buds of the first and second primary scales and axillary buds of cotyledons of 4 to 6 day old pea seedlings. Meristem explants maintained a high proliferation ability in each subculture in the course of 20 months of the culture. Regenerated shoots were rooted in the same basal medium containing 5 M NAA. Rooted plants were cultured in hydroponic pots filled with half-strength MS-medium to attain anthesis and seed maturity. The phenotypic uniformity of the regenerants was evaluated. Cytological investigation confirmed the diploid stage (2n=14) of regenerants and their progeny. Histological studies revealed that proliferating shoots originated from axillary and adventitious buds. In vitro propagation is discussed as related to pea breeding.     

Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium

Summary Derivatives of potato (Solanum tuberosum cv.'s Maris Bard and Desiree) transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixedcultures of shoot-inducing A. tumefaciens strains T37 and either Agrobacterium strain LBA1834(pRAL1834), (Hille et al. 1983) or LBA4404(pBIN6; pRAL4404), (Bevan 1984). Two small-scale feasibility experiments gave at least four Maris Bard plants transformed with pRAL1834 T-DNA and two Desiree plants with pBIN6 T-DNA. The transformed Maris Bard plants were morphologically abnormal and highly aneuploid. This was probably an unfortunate side-effect of a tissue culture-step introduced to promote the efficiency of shoot regeneration. The transformed Desiree plants, in contrast, were isolated without promoting additional shoot-growth. They were morphologically normal, contained 47 and the euploid 48 chromosomes per cell respectively and had improved growth on media containing kanamycin.     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Regeneration of the tropical legume Aeschynomene sensitiva Sw. from root explants

Regeneration of Aeschynomene sensitiva Sw. after callogenesis was obtained from small (2–5 mm long) root explants of 30-day-old seedlings aseptically cultivated on Murashige and Skoog medium supplemented with various concentrations of growth regulators. After 4 weeks, the best results were observed with 0.54 M -naphthaleneacetic acid and 2.22 M benzyladenine. On this medium, the rate of regeneration depended on seedling age and agar concentration. The highest number of shoots per explant was obtained with small cuttings from 30-day-old seedlings grown on a medium containing 8 g l^–1 of agar. Regeneration success was also dependent on explant size. When longer explants (7–20 mm) were cut from the main root, direct regeneration was obtained in two weeks. These cuttings also generated shoots through callogenesis in four weeks but always in lower quantities than with direct regeneration, whatever the seedling age. here also, the best regeneration was obtained with cuttings from 30-day-old seedlings maintained on a medium with 8 g l^–1 of agar. Regenerants were rooted on growth-regulator-free Murashige and Skoog medium and then acclimatized in a greenhouse. A better survival to transplantation was observed when plantlets were inoculated with the photosynthetic Bradyrhizobium strain ORS 278. Stem and root nodules developed on the inoculated plantlets and were able to fix nitrogen.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA - naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Adventitious shoot production from calloid cultures of banana

Isolated tips (approx. 2 mm long) from aseptic, multiplying shoot cultures of the triploid dessert banana clone Highgate were tested for their morphogenetic responsiveness to hormone treatments on semisolid media. Medium containing Murashige and Skoog (1962) salts, p-chlorophenoxyacetic acid, and kinetin produced a compact calloid mass. Protuberances disclosed by SEM as rounded, button-shaped, and pointed outgrowths resembling fasciated shoots were formed in profusion. Sections showed many meristematic regions, some associated with distinct leaf primordia. Formation and growth of successive leaves yielded small, elongated, adventitious shoots with constricted bases. Transferral to a basal MS medium with 1 mg/l 1-naphthaleneacetic acid (NAA) led to the formation of rooted plantlets.     

Shoot regeneration from petioles and leaves of Vitis X labruscana ‘Catawba’

Summary Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana Catawba. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.0–10.0 M BA and 0.1–0.5 M IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NN69 Nitsch and Nitsch (1969) medium - NOA 2-naphthoxyacetic acid     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench)

Anther culture for haploid induction of buckwheat was studied over a period of five years. Approximately 24,000 anthers were isolated and cultured on different culture media. The regeneration capacity was generally very low. Data are presented for experiments that included 7278 anthers on which 99 calluses were formed and 20 buds regenerations were noted. Regeneration occurred most readily on gellan-gum solidified media, with 90 g l^-1 maltose, 2.5 mg l^-1 BA, 0.5 mg l^-1 IAA, and preferably in darkness. Haploid cells, as established by chromosome counts, were observed in eight regenerants. Several abnormalities of pollen development in vitro were detected. Starch presence in pollen as a possible sign of androgenic capacity was studied. Microspores in uninucleate and early binucleate stages contained only proplastids, while in adult pollen grains a number of amyloplasts were present.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - 2,4 D-2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - 2iP 6- c,c-dimethylallylaminol-purine - NAA -naphthalene acetic acid     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.