Plant tissue culture has become one of the fundamental tools of plant science research. It is extensively employed in the production, conservation and improvement of plant resources. The presence of somaclonal variation in populations derived from tissue culture is affecting the use of tissue culture negatively and has remained a major problem. Conversely, it is a source of new desirable clones/variants with better agronomic traits. In this review, we summarize the possible causes, detection methods and desirability of variants. Somaclonal variation is one of the most researched and reviewed topics. Hence, we restricted ourselves to outlining various examples which may be used as important references for researchers who intend to identify and/or characterize somaclonal variants while using tissue culture for research and production. Emphasis is placed on the negative effects of somaclonal variation. However, this review also includes examples of some useful variants generated as a result of somaclonal variation.

Somaclonal variation in plants: causes and detection methods

The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed.

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Somaclonal variations and their applications in horticultural crops improvement

Tissue culture of ornamental pot plant: A critical review on present scenario and future prospects

The components of plant tissue culture media II. Organic additions, osmotic and pH effects, and support systems

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.

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Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Somatic embryos were regenerated in vitro from calamondin style–stigma explants cultured in the presence of N6-benzylaminopurine (BAP) cytokinin and three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), N-phenyl-N′-benzothiazol-6-ylurea (PBU) and N,N′-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU). The phenylurea derivative compounds tested at micromolar level (12 μM) were able to induce a percentage of responsive explants significantly higher from that obtained with BAP and hormone-free (HF) conditions. In order to verify the genetic stability of the regenerants, 27 plants coming from different embryogenic events were randomly selected from each different culture condition and evaluated for somaclonal variations using inter-simple sequence repeat and random amplified polymorphic DNA analyses. We observed that 2,3-MDPU and PBU gave 3.7% of somaclonal mutants, whereas 4-CPPU gave 7.4% of mutants. No somaclonal variability was observed when plantlets were regenerated in BAP or HF medium. Although diphenylurea derivatives show a higher embryogenic potential as compared to BAP, they induce higher levels of somaclonal variability. This finding should be taken in consideration when new protocols for clonal propagation are being developed.

Genetic instability in calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives.

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L) Osb] from cultures of stigma/style explants and undeveloped ovules Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 46 μM kinetin; and 500 mg l-1 malt extract and 133 μM 6-benzylaminopurine Sucrose (146 mM) was used as carbon source Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract Plants were successfully transferred to soil

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Callus induction, somatic embryogenesis and plant regeneration were obtained in lemon [Citrus limon (L.) Burm. cv `Femminello'] and sweet orange [C. sinensis (L.) Osb. cv `Washington Navel GS'] from cultures of stigma and style transverse thin cell layer explants [(t)TCLs]. Explants were cultured on 16 different media, based on the nutrients and vitamins of Murashige and Tucker medium (MT) supplemented with different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU). Sucrose (146 mM) was used as the sole carbon source. Somatic embryos arose from callus at the surface of stigma and style (t)TCLs 3–5 months after culture initiation of both sweet orange and lemon. The percentages of embryo formation from style (t)TCLs ranged from 0% (the media containing 2,4-D) to 16.0% (the medium supplemented with 4 μM 4-CPPU) for C. limon. Better results were obtained when stigma (t)TCLs from C. limon were used; in fact, percentages ranged from 0% on the media containing 2,4-D, with the only exception for the medium supplemented with 0.4 μM 2,4-D, to 24.8% on medium with 4 μM 4-CPPU. The embryogenic response of lemon (t)TCLs was usually higher than for sweet orange (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies ranging from 53% to 75% for sweet orange and lemon style derived embryos, respectively.

Effect of 2,4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus

Somatic embryogenesis in Citrus from styles culture

The genus Citrus is cultured in more than 100 countries making it one of the most important commercial fruit crops in terms of economic value and human nutrition (Barlass and Skene, 1986). Fresh fruits and juice are the most significant citrus products, but essential oils, pectin, and marmalade, as well as candied and dried rinds, also have commercial value (Barlass and Skene, 1986). In recent years, the importance of Citrus for the production of ornamental plants has increased considerably (De Pasquale and Carimi, 1998).

Fabio De Pasquale

Papers

Genetic instability in calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives.

Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Effect of 2,4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus

Somatic embryogenesis in Citrus from styles culture

Micropropagation of Citrus