RAPD and phenotypic analysis were conducted to assess clonal stability of hazelnuts generated from axillary buds cultured in vitro for long-term. The nuts produced on in vitro-propagated plants were indistinguishable from those of donor plants. With the exception of rare horizontal (plagiotropic) growth, all in vitro-propagated plants exhibited phenotypes similar to those of donor plants. RAPD analysis did not reveal any somaclonal variation between donor plants from which in vitro cultures were initiated and micropropagated plants (6-year cultures), and no somaclonal variation was detected among in vitro-propagated plants. However, polymorphism (15.6%) was detected between the parent plant and its in vitro-propagated progenies (from seedlings). These results show a good discriminatory power of RAPD to detect polymorphism between samples where it is expected, and it can be effectively used for genetic assessment of micropropagated hazelnut. No evidence of genetic or epigenetic changes was observed in long-term cultured hazelnut, and thus long-term in vitro culture of hazelnut does not seem to limit its clonal propagation.
Mehmet Nuri Nas, Nedim Mutlu and Paul E. Read
M.A. Norton and R.M. Skirvin
Chimeral `Thornless Evergreen' (CTE), (Rubus laciniatus Willd.) somaclones selected in 1983 and field planted in 1985 were reexamined in 1992 for various vegetative and reproductive characteristics. Two major types of thornless (prickle-free) plants, intermediate-sized (`UI 6-6' = `Everthornless') and dwarf (`UI 6-4'), originally selected from a chimeral thornless parent plant, were compared with thorny plants. The intermediate and dwarf somaclones have maintained their distinctive habits over 7 years' growth in the field, indicating that their growth habits are stable and not a transient effect of tissue culture. Although the thornless somaclones remained thornless, the degree and type of prickle-like structures varies considerably, indicating that the thornless gene (S te) does not entirely suppress the production of prickles, but apparently alters their development. Increasing suppression was directly related to increasing dwarfism, suggesting a link between thornlessness and internode length.
Frederic Ngezahayo, Wanli Guo, Lei Gong, Fangxia Li, Bao Liu and Yingshan Dong
The authors have previously reported an efficient in vitro system for mass micropropagation of Robinia ambigua `idahoensis' (Idaho locust). Their method used enhanced branching of axillary buds from a single donor plant along with detection of somaclonal variation by the intersimple sequence repeat (ISSR) markers. Because ISSRs tend to be clustered to specific chromosomal regions in plant genomes, the extent and scope of the genomic variations and the sequences underlying the variation warranted further investigations. In this study, the authors analyzed the same set of 40 randomly selected micropropagated R. ambigua plants by a more general molecular marker—random amplified polymorphic DNA (RAPD) using 34 selected primers. In addition, they sequenced some of the variable bands. Of the 260 reproducible RAPD bands scored, 70 were polymorphic among the 41 plants (40 micropropagated and one donor), corresponding to a polymorphism level of 26%. Cluster analysis revealed a genetic similarity ranging from 0.61 to 0.97. Of the 20 sequenced bands underlying the variations, eight showed significant homology to known or predicted functional cellular genes or retroelements. These results clearly indicated that micropropagation of R. ambigua, even by enhanced branching of axillary buds, can be accompanied by extensive genomic variations, which should be taken into account for commercial propagation of this plant by in vitro means.
Hamidou F. Sakhanokho and M. Nurul Islam-Faridi
microscope field of view (per 92,792.53-μm 2 area), and stomatal density (per 0.00092793-cm 2 area). Somaclonal variation is variation in regenerated plants that takes place as a result of tissue culture of any type, although some scientists require that
Shelley Jansky, Sandra Austin-Phillips and Corine McCarthy
The Colorado potato beetle (CPB) is a major insect pest that is controlled mainly through the use of pesticides. Development of potato clones with multiple forms of host plant resistance may provide a stable alternative or supplemental form of CPB control. Tetraploid hybrids were developed by somatic fusion of diploid interspecific Solanum clones with different forms of resistance to CPB. Hybrids were created between a clone containing leptine glycoalkaloids and four clones producing glandular trichomes. One fusion produced vigorous hybrids that were analyzed for CPB resistance traits. Somaclonal variation among hybrids was detected for trichome density and resistance to feeding by adult and larval beetles. Somatic hybrids were less resistant than the parents in adult feeding preference trials, but several were more resistant than either parent in larval feeding trials. Future studies are needed to determine whether clones producing both glandular trichomes and leptines express resistance that is more stable than that of clones with only one resistance factor.
Carol Gonsalves, Baodi Xue, Marcela Yepes, Marc Fuchs, Kaishu Ling, Shigetou Namba, Paula Chee, Jerry L. Slightom and Dennis Gonsalves
A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.
M.J. Prado, M.T. Herrera, R.A. Vázquez, S. Romo and M.V. González
A simple and reliable protocol for micropropagation during 12 subcultures of two field growth male plants of kiwifruit [Actinidia deliciosa (A.Chev.) Liang and Ferguson] is described. The best results of shoot multiplication and elongation were obtained in Cheng's K(h) medium in the presence of 0.5 μm NAA, 22 μm BA and 1.4 μm GA3 for `Tomuri' explants, and of 0.1 μm NAA, 4.4 μm BA, and 0.3 μm GA3 for clone A explants. In addition, the cytokinin compounds TDZ and mT were also tested allowing improving the multiplication rate in `Tomuri' explants. For rooting, `Tomuri' and clone A developed shoots were treated by basal immersion in a 5 mm IBA solution for 15 seconds. Treated shoots were then cultured in half-strength K(h) medium without growth regulators showing 100% rooting after 30 days. Regenerated plantlets were successfully transplanted to soil (90% survival) and they are actively growing in the field. Somaclonal variation analysis by AFLP was carried out using 15 primer combinations, yielding reproducible and well-resolved bands with a 57% of polymorphism. AFLP markers showed to be effective to discriminate genetic variation in this species, being greater in clone A than `Tomuri'. Chemical names used: N6-benzyladenine (BA); gibberellic acid (GA3); indole-3-butyric acid (IBA); meta-topolin (mT); naphthaleneacetic acid (NAA); thidiazuron (TDZ).
Vyacheslav Gurevich, Uri Lavi and Yuval Cohen
Date palm (Phoenix dactylifera L.) is a major tree crop in arid regions of the Middle East and North Africa, having an important impact on the economy of many countries in these regions. Date palms are traditionally propagated through offshoots. The development of propagation methods through tissue culture resulted in massive expansion of date palm plantations. While most trees generated from tissue culture are normal and true-to-type, several typical abnormal phenotypes are detected. The present study applies amplification fragment length polymorphism (AFLP) analysis to characterize the genetic variation of two elite date cultivars, `Barhee' and `Medjool', as well as male clones, propagated from offshoots and through tissue culture. The two cultivars have very distinct AFLP band patterns. Most offshoots, as well as the tissue culture-propagated plants, have very similar band patterns, demonstrating a low level of genetic variation. However, a significant level of genetic variation was detected among `Medjool' plants generated from tissue culture. Several phenotypically abnormal trees were characterized by unique and different AFLP band patterns. The male clones are characterized by a high level of polymorphic bands. Genetic variation was also detected between various tissues of variegated `Medjool' trees propagated from tissue culture. The significance of these results, regarding the mechanism of the phenomenon and its relevance to agricultural practice, is discussed.
Patrick P. Moore, Jo Ann Robbins and Thomas M. Sjulin
The field performance of micropropagated and runner-propagated subclones of `Olympus' strawberry (Fragaria × ananassa Duch.) was compared. The yield of micropropagated plants was not greater than that of runner-propagated plants. There was significant variability among micropropagated subclones, with the highest yielding subclone having 68% higher yield than the lowest yielding subclone in each of the first 2 years. However, after runner propagation for 4 years, selected subclones showed no differences in yield. Differences among subclones of `Olympus' were not stable and were most likely transient responses to the micropropagation environment. The apparent superiority of the subclones was not genetic.