Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1–2 mm diameter colonies were formed in 5–6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l−1 each of 2,4-D, BA and KN and 500 mg l−1 CH for further growth. Once the colonies had become green, compact and nodular, and were 8–10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8–12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.

Regeneration of fertile plants from protoplasts of soybean (Glycine max L. Merr.): genotypic differences in culture response.

A procedure for the preparation of tomato chromosome suspensions suitable for flow cytometric analysis is described. Rapidly growing cell suspension cultures of Lycopersicon esculentum cv VFNT cherry and L. pennellii LA716 were treated with colchicine to enrich for metaphase chromosomes. Metaphase indices between 20 and 35% were routinely obtained when cultures were exposed to 0.1% colchicine for 15–18 h after 2 days of subculture. Mitotic cells were isolated by brief treatment with cell wall digesting enzymes in a medium with low osmolarity (∼325 mOsm/kg of H52O). The low osmolarity medium was needed to avoid the chromosome clumping and decondensation seen in standard media. Suspensions of intact chromosomes were prepared by lysing swollen protoplasts in various buffers (MgSO4, polyamines, hexylene glycol, or KCl-propidium iodide) similar in contents to the buffers used to isolate mammalian chromosomes. For univariate flow cytometric analysis, chromosome suspensions were stained with a fluorescent DNA-binding stain (propidium iodide, Hoechst 33258, mithramycin, or chromomycin A3) and analyzed using an EPICS flow cytometer (Profile Analyzer or 753). Peaks for the chromosomes, chromatids, clumps of chromosomes, nuclei, and fluorescent debris were seen on a histogram of log of fluorescence intensity, and were confirmed by microscopic examination of the objects collected by flow-sorting. Chromosome suspensions prepared in MgSO4 buffer have the highest frequency of intact chromosomes and the least fluorescent cellular debris. Peaks similar to theoretical univariate flow karyotypes of tomato chromosomes were seen on the observed univariate flow karyotypes, but were not as well resolved. Bivariate flow analysis of tomato chromosome suspension using double-stain combination, Hoechst 33258 and chromomycin A3, and two laser beams showed better resolution of some chromosomes.

Preparation and flow cytometric analysis of metaphase chromosomes of tomato.

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

/pdf/tomato-a-crop-species-amenable-to-improvement-by-cellular-46d073uz2b.pdf

Tomato: a crop species amenable to improvement by cellular and molecular methods

Asymmetric somatic hybrids were recovered following fusion of tomato leaf mesophyll protoplasts with irradiated protoplasts isolated from Lycopersicon pennellii suspension cells. The asymmetry was determined by scoring the regenerants at between 20 and 24 loci using isozymes and restriction fragment length polymorphisms. In addition, three quantitative traits, fruit size, leaf shape, and stigma exsertion, were measured in the regenerants. The recovery of asymmetric somatic hybrids was as high as 50% of the regenerants, and there was no requirement for the transfer of a selectable marker gene from the irradiated partner. The amount of nuclear DNA transferred from the irradiated protoplast fusion partner was found to be inversely proportional to the radiation dose. It was possible to recover tomato asymmetric somatic hybrids which were self-fertile and contained limited amounts of genetic information from L. pennelli.

Effect of radiation dose on the production of and the extent of asymmetry in tomato asymmetric somatic hybrids.

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 μM concentrations. Direct shoot formation occurred on media with 10 μM kinetin and 0.001 μM IAA or NAA. Root formation was observed on media with 0.1–10 μM IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 μM IAA and 0.1 μM kinetin. Shoot organogenesis was increased by substituting 10 μM zeatin or N6-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (‘Red Alert’) to 5.3 (‘Large Red Cherry’). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.

Shoot, root and flower morphogenesis on tomato inflorescence explants

Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 μl droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).

Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration.

E. M. Rietveld

Papers

Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration.

Regeneration of leaf mesophyll protoplast of tomato cultivars.