In vitro chromosome doubling can be induced by several antimitotic agents. The most commonly used are colchicine, oryzalin and trifluralin. The process of induced chromosome doubling in vitro consists of a typical succession of sub-processes, including an induction phase and a confirmation protocol to measure the rate of success. The induction step depends on a large number of variables: media, antimitotic agents, explant types, exposure times and concentrations. Flow cytometry is the pre-eminent method for evaluation of the induced polyploidization. However, alternative confirmation methods, such as chromosome counts and morphological observations, are also used. Since polyploidization has many consequences for plant growth and development, chromosome doubling has been intensively studied over the years and has found its way to several applications in plant breeding. This review gives an overview of the common methods of chromosome doubling in vitro, the history of the technique, and progress made over the years. The applications of chromosome doubling in a broader context are also discussed.

Mitotic chromosome doubling of plant tissues in vitro

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.

Initiation, growth and cryopreservation of plant cell suspension cultures

Polyploid Spathiphyllum wallisii Regel 'Speedy' plants were obtained from somatic embryos induced on anther filaments exposed to mitosis inhibitors. Primary embryos yielded less polyploid plantlets than secondary embryos. Colchicine could be efficiently replaced by oryzalin or trifluralin (10 µM), resulting in an average yield of 5% polyploids. The mitosis inhibitors were directly added to the induction medium. Morphological differences between diploid and tetraploid S. wallisii hybrids were observed. Throughout our experiments flow cytometry was applied as an unambiguous screening tool. Ploidy breeding within the genus Spathiphyllum holds promises towards the development of tetraploid hybrids with an altered morphology, or triploids with a reduced fertility.

Chemically induced polyploidization in Spathiphyllum wallisii Regel through somatic embryogenesis

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.

/pdf/cryopreservation-of-protocorm-like-bodies-plbs-of-10aayx0qcs.pdf

Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulation-dehydration

In vitro regeneration systems provide a powerful tool for manipulating ploidy to facilitate breeding and development of new crops. Polyploid induction can expand breeding opportunities, assist with the development of seedless triploid cultivars, enhance ornamental characteristics and environmental tolerances, increase biomass and restore fertility in wide hybrids. In vitro ploidy manipulation is commonly induced using antimitotic agents such as colchicine, oryzalin and trifluralin, while many other antimitotic agents have been relatively unexplored. Successful induction requires a synergistic pairing of efficient penetration of the antimitotic agent and may be dependent the length of exposure and concentrations of antimitotic agents, tissue types, and interactions with basal media and plant growth regulators. In vitro conditions vary among taxa and individual genera, species, and cultivars, often requiring unique treatments to maximize polyploid induction. In some taxa, the induction of polyploidy influences in vitro growth, development, and root formation. Here we provide an overview of mitotic inhibitors and their application for in vitro ploidy manipulation for plant breeding and crop improvement.

/pdf/in-vitro-ploidy-manipulation-for-crop-improvement-co0pjs6a32.pdf

In vitro Ploidy Manipulation for Crop Improvement.

Preservation of plant germplasm is important to safeguard biodiversity and to store elite plants. Cryopreservation is one of the possible preservation techniques. Research for a cryopreservation protocol is often inefficient because of slow or poor regrowth of plant material. Therefore, at least one technique, that allows a quick and accurate prognosis of viability after cryopreservation, is required. We evaluated five techniques: electrolyte leakage, triphe-nyltetrazoliumchloride (TTC) staining (visual and spectrophotometrical analysis), malondialdehyde concentrations in plant tissue and a mathematical model that relates ‘water content’ to the weight of encapsulated plant material. Electrolyte leakage and TTC-staining (if visually analysed) are efficient to predict viability. Our mathematical model allows us to save time and plant material in order to develop an efficient encapsulation—dehydration protocol. All other techniques were rejected because of the high variability of the results. This is due to the variability of biochemical activity in plant tissue and the small amount of tissue used in the experiments.

Evaluation of analytical techniques to predict viability after cryopreservation

In vitro polyploidy induction offers the possibility of quick multiplication over in vivo methods, and is therefore particularly interesting for slowly growing plants. Three different in vitro protocols for the induction of tetraploid Rhododendron simsii hybrids were evaluated. The most successful technique was a daily application of mitotic poison on the apical meristem of seedlings. However, this technique was quite labour intensive. Direct sowing on media enriched with antimitotic agents requires less work but is less efficient. To induce ploidy chimeras starting from an existing genotype, mitotic inhibitors can be added to multiplication media. Abbreviations COL (colchicine), DAPI (4,6-diamidino-2-phenylindol), LSD (least significant difference), ORY (oryzalin), PAR (photosynthetic active radiation), TRI (trifluralin)

In vitro polyploidy induction in Rhododendron simsii hybrids.

/pdf/in-vitro-polyploidy-induction-in-rhododendron-simsii-hybrids-2ve8zduhjf.pdf

Shoots can be regenerated from leaf explants of most azalea (Rhododendron simsii) cultivars using a two-step protocol with a callus induction and a regeneration phase. This procedure is unsuccessful for a few cultivars, because of excessive callus production. In most such cases, shoot regeneration could be obtained in one step, starting from petioles as explants. Flower colour variations (the induction of sports) were frequently observed after regeneration. This phenomenon offers prospects for the enlargement of the commercial assortment but can also be used for the study of gene regulation processes in flower colour variegation.

Adventitious shoot regeneration and appearance of sports in several azalea cultivars

To introduce yellow, blue or orange pigmentation in the Rhododendron subgenus Tsutsusi (including R. simsii hybrids) by classical breeding, intersubgeneric pollination experiments involving Hymenanthes, Pentanthera and Rhododendron (including Vireya) species and hybrids were started. Prefertilization research by pollen tube staining revealed a different behaviour between bilateral crosses. Ovule culture was applied as a means to save a substantially larger amount of hybrid embryos from abortion. Though flower/seed capsule abortion eliminated many crosses in an early developmental stage, ovules of many crosses could be initiated in vitro on various time intervals after pollination. Germination was achieved on basal Rhododendron medium +50 mg/l GA(3). Green seedlings were obtained after Tsutsusi x Hymenanthes, Hymenanthes x Tsutsusi, Tsutsusi x Rhododendron, Rhododendron x Tsutsusi and Vireya x Tsutsusi pollination. Many seedlings have little growth vigour and next to green seedlings albino seedlings occur frequently, depending on the pollination type. The first true leaves of many assumed hybrids obviously lack chlorophyll. The ploidy level of Vireya x Tsutsusi and Tsutsusi x Pentanthera seedlings is intermediate, which strongly suggests their hybrid origin.

G. Samyn

Papers

Evaluation of analytical techniques to predict viability after cryopreservation

In vitro polyploidy induction in Rhododendron simsii hybrids.

In vitro polyploidy induction in Rhododendron simsii hybrids.

Adventitious shoot regeneration and appearance of sports in several azalea cultivars

Interspecific breeding in the Rhododendron genus involving R. simsii hybrids