In Vitro Selection

Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in almost every country of the world. Development of protocols for in vitro selection can provide new advances for the production of stress tolerant cultivars. Techniques have been optimised for the production of haploids and somatic hybrids. Attempts have also been made to transfer the higher regenerative ability of wild varieties to cultivated tomatoes. Although, some information is available on the morphogenesis of tomato, the techniques have not been developed to a level at which they can be utilised in large-scale multiplication of commercially important cultivars. The morphogenesis response seems to be highly dependent PGRs used in the media, which is again cultivar and genotypic specific. Somatic embryogenesis in tomato is still at its infancy, and efficient procedures for large-scale production via somatic embryogenesis are yet to be developed. Genetic stability of the tissue culture raised tomato plants also needs to be addressed. The use of a combination of molecular and conventional breeding techniques could be the option for the development of cultivars resistant to biotic and abiotic stresses. This paper reviews the advances made in various aspects of tissue culture in tomato. It also discusses the issues that still need to be addressed to utilise the full potential of plant tissue culture techniques in genetic improvement and mass propagation of tomato.

The exploration of somaclonal variation is an approach that could provide date palm breeding programs with new genotypes. Naturally occurring or induced variants may have superior agronomic quality and/or enhanced performance but could also harbor new traits such as tolerance to drought and salinity or resistance to major diseases i.e. bayoud. This chapter summarizes recent progress in terms of studying and exploring date palm somaclonal variation, and provides an outlook about future applications of this biotechnology in this socioeconomically important crop.

Wilt caused by Fusarium oxysporum f. sp. pisi is a serious production constraint for peas worldwide. An attempt was made to isolate wilt-resistant mutants in two susceptible pea genotypes, Arkel and Azad P-1, employing induced mutagenesis and in vitro selection techniques. Two thousand seeds of each genotype were mutagenized either with ethyl methane sulfonate (EMS, 0.2% and 0.3%) or gamma rays (5-22.5 kR) in 60Co gamma cell for three consecutive years. Screening of different mutagenized populations under wilt-sick plots resulted in the isolation of 25 mutants exhibiting complete or enhanced wilt resistance compared to parental genotypes. Five of these wilt-resistant mutants also outperformed the susceptible background genotypes in terms of yield and other horticultural traits. Efforts were also made to isolate wilt-resistant regenerants from callus cultures exhibiting insensitivity to culture filtrate (CF) of F. oxysporum f. sp. pisi. A total of 250 regenerants (R 0) were obtained from CF-insensitive calli selected from medium supplemented with 20% culture filtrate. When evaluated in artificially inoculated sick plots, only five R 2 lines obtained from the regenerants exhibited enhanced wilt resistance compared to parental cultivars. However, the selected lines did not exhibit resistance levels equivalent to those shown by wilt-resistant lines isolated through in vivo mutagenesis. To conclude, induced mutagenesis through irradiation and EMS treatments exhibited superiority over in vitro selection for inducing wilt resistance in peas.

Glioblastoma multiforme is the most malignant form of brain tumor. Despite treatment including surgical resection, adjuvant chemotherapy, and radiation, these tumors typically recur. The recurrent tumor is often resistant to further therapy with the same agent, suggesting that the surviving cells that repopulate the tumor mass have an intrinsic genetic advantage. We previously demonstrated that cells selected for resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) are near-diploid, with over-representation of part or all of chromosomes 7 and 22. While cells from untreated gliomas often have over-representation of chromosome 7, chromosome 22 is typically under-represented. We have analyzed cells from primary and recurrent tumors from the same patient before and after in vitro selection for resistance to clinically relevant doses of BCNU. Karyotypic analyses were done to demonstrate the genetic makeup of these cells, and fluorescent in situ hybridization analyses have defined th...

As part of an ongoing research project on biological control of apple scab, this study presents a novel approach for the in vitro selection of potential antagonists of the saprophytic phase of Venturia inaequalis. A collection of forty-two fungal isolates were tested for their in vitro ability to degrade apple leaf tissue, inhibit pseudothecia, and ascospore production. The inhibition of ascospore production cannot always be linked reliably with leaf degradation or the evaluation of pseudothecia production. Consequently, ascospore production was retained as the most useful screening parameter. Six isolates proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of fungal and bacterial saprophytes can now be based on a reliable and simple in vitro screening method.

Significant differences were found be
tween tomato genotypes for their ab
ility for callus formation on the two
types of explants on the different types of media. Th
e highest genotype for callus formation was Super Marmand
(77.10%) and the lowest genotype for callus formation was 12 M (56.3 %). M
2
medium (83%) could be recommended
as appropriate medium for callus induction for all genotypes.
Concerning the type of explants the results showed that
cotyledon leaf explants (70.39%) were better than hypocotyl
s explants (66.67%) for callus formation. In the present
experiment the mean numbers of regenerated shoots s
howed highly significant differences between genotypes.
Amcostar exhibited large number of shoots under all factors studied as compared to other genotypes. The effect of
genotypes and media interaction on plant regeneration from
callus was highly significant. The best combination for
indirect regeneration was Amcostar genotype on M
4
medium (93.8% with a mean 9.7). The growth dynamic of callus
tissue was evaluated during the growing period of 30 days on the basic medium MS supplemented with different
concentration of PEG, The concentrations of PEG in the medium showed a great effect on the growth value at the end
of 30 days growing period.  The mass of callus and shoots re
generated directly from explants were evaluated after 60
days of growing on regeneration MS medium supplemented
with different concentration of PEG. The highest dry
weight was achieved when the Peto-86
explants were cultivated on MS supplem
ented with 75.00 gram PEG.  Shoots
regeneration frequency of cotyledon segment of tomato
genotype ranged from 12.00 to 82.40 %. The highest shoot
regeneration was achieved on the explants
of Super Strain B it was 2.14 shoot pe
r explants. The mean number of shoots
per explants was decreased by increasing the concentration of mannitol in the medium the mean numbers of shoots per
explants were 1.811, 1.59, 1.50, 1.34 and 1.43 for the Ma
nnitol concentration 0.0, 25.0, 50.0, 75.0 and 100.0 mM/L,
respectively. The regeneration capability
it's self-affected strongly by the in
creasing of mannitol in the medium.

A DNA enzyme isolated via in vitro selection can be used to cleave essentially any RNA substrate in the presence of a divalent metal cation, preferably Mg2+, provided that the substrate has a purine-pyrymidine junction. Practical applications exist, as some viral genomes are made ...

The possibility of using in vitro shoot apex culture to evaluate salt tolerance of cultivated (Lycopersicon esculentum Mill.) and wild (Lycopersicon pennellii (Correll) D'Arcy) tomato species was determined and related to the response obtained by callus culture. Both apices and calluses were grown on media supplemented with 0, 35, 70, 105, 140, 175 and 210 mM NaCl, and growth and physiological traits were determined. Most apices of L. esculentum did not develop roots from low NaCl levels, whereas the apices of L. pennellii were able to develop roots at the different salt levels. This different degree of salt tolerance between L. esculentum and L. pennellii was not, however, clearly shown on the basis of the shoot growth of the plantlets. The callus response was similar to that shown by the rooting parameters, as callus growth in response to increased salinity was much greater in L. pennellii than in the tomato cultivar. K+decreased more and proline accumulated less with salinity in shoots of L. esculentum compared to L. pennellii, whereas the opposite response was obtained in calluses. The results obtained in this study suggest that rooting parameters are the most useful traits for rapid evaluation and screening of tomato species and segregating populations through in vitro shoot apex culture.

Nucleolin, a multi-domain protein involved in ribosome biogenesis, has been shown to bind the consensus sequence (U/G)CCCG(A/G) in the context of a hairpin loop structure (nucleolin recognition element; NRE). Previous studies have shown that the first two RNA-binding domains in nucleolin (RBD12) are responsible for the interaction with the in vitro selected NRE (sNRE). We have previously reported the structures of nucleolin RBD12, sNRE and nucleolin RBD12-sNRE complex. A comparison of free and bound sNRE shows that the NRE loop becomes structured upon binding. From this observation, we hypothesized that the disordered hairpin loop of sNRE facilitates conformational rearrangements when the protein binds. Here, we show that nucleolin RBD12 is also sufficient for sequence- specific binding of two NRE sequences found in pre-rRNA, b1NRE and b2NRE. Structural investigations of the free NREs using NMR spectroscopy show that the b1NRE loop is conformationally heterogeneous, while the b2NRE ...

Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in almost every country of the world. Development of protocols for in vitro selection can provide new advances for the production of stress tolerant cultivars. Techniques have been optimised for the production of haploids and somatic hybrids. Attempts have also been made to transfer the higher regenerative ability of wild varieties to cultivated tomatoes. Although, some information is available on the morphogenesis of tomato, the techniques have not been developed to a level at which they can be utilised in large-scale multiplication of commercially important cultivars. The morphogenesis response seems to be highly dependent PGRs used in the media, which is again cultivar and genotypic specific. Somatic embryogenesis in tomato is still at its infancy, and efficient procedures for large-scale production via somatic embryogenesis are yet to be developed. Genetic stability of the tissue culture raised tomato plants also needs to be addressed. The use of a combination of molecular and conventional breeding techniques could be the option for the development of cultivars resistant to biotic and abiotic stresses. This paper reviews the advances made in various aspects of tissue culture in tomato. It also discusses the issues that still need to be addressed to utilise the full potential of plant tissue culture techniques in genetic improvement and mass propagation of tomato.