Anne Kathrine Hvoslef-Eide

Liquid culture systems for in vitro plant propagation

Contributing Authors Preface 1. General introduction: a personal reflection on the use of liquid media for in vitro culture Walter Preil I. Bioreactors 2. Application of bioreactor design principles to plant micropropagation Wayne R. Curtis 3. Bioreactor design for propagation of somatic embryos Anne Kathrine Hvoslef-Eide, Odd Arild S. Olsen, Ragnhild Lyngved, Cristel Munster & Petter H. Heyerdahl 4. Practical aspects of bioreactor application in mass propagation of plants S. Takayama & M. Akita 5. Simple bioreactors for mass propagation of plants Meira Ziv 6. Application of bioreactor systems for large scale production of horticultural and medicinal plants K.Y. Paek, Debasis Chakrabarty & E.J. Hahn 7. Membranes to reduce adherence of somatic embryos to the cell lift impeller of a bioreactor Seppo Sorvari, Riitta Makelainen, Katriina Ahanen & Otto Toldi 8. Cost-effective mass cloning of plants in liquid media using a novel growtek bioreactor Satyahari Dey 9. Multiplication of Chrysanthemum shoots in bioreactors as affected by culture method and inoculation density of single node stems Eun-Joo Hahn & Kee-Yoeup Paek 10. Control of growth and differentiation of bioreactor cultures of Physcomitrella by environmental parameters Annette Hohe & Ralf Reski II. Temporary Immersion Systems 11. Temporary immersion system: a new concept for use liquid medium in mass propagation M. Berthouly & H. Etienne 12. Mass propagation of tropical crops in temporary immersion systems Elio Jimenez Gonzalez 13. Use of growth retardants for banana (Musa AAA cv. Grand Naine) shoot multiplication in temporary immersion systems Nilca Albany, Elio Jimenez, Jorge Vilchez, Leyanis Garcia, Manuel De Feria, Naivy Perez, Zoe Sarria, Blanca Perez & Justo Clavelo 14. Somatic embryogermination of Psidium guajava L. in the Rita(R) temporary immersion system and on semisolid medium Rafael Gomez Kosky, J. Vilchez Perozo, N. Albany Valero & D. Agramonte Penalver 15. Application of a temporary immersion system in mass propagation of Phalaenopsis Tino Hempfling & Walter Preil 16. Propagation of Prunus and Malus by temporary immersion C. Damiano, S.R. La Starza, S. Monticelli, A. Gentile, E. Caboni & A. Frattarelli 17. Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle Li-Hua Zhu, Xue-Yuan Li & Margareta Welander 18. Experimental use of a novel temporary immersion system for liquid culture of olive microshoots Katerina Grigoriadou, Miltiadis Vasilakakis, Theofilos Tzoulis & Eleftherios P. Eleftheriou 19. Shoot regeneration from nodules of Charybdis sp.: A comparison of semisolid, liquid and temporary immersion culture systems Ch. Wawrosch, A. Kongbangkerd, A. Kopf & B. Kopp III. Somatic Embryogenesis And Shoot Initiation 20. Propagation of Norway spruce via somatic embryogenesis Sara von Arnold, Peter Bozhkov, David Clapham, Julia Dyachok, Lada Filonova, Karl-Anders Hogberg, Mathieu Ingouff & Malgorzata Wiweger 21. Norway spruce somatic embryogenesis: membrane rafts as a compromise between liquid and solidified media M. Vagner, Z. Vondrakova, L. Fischerova & J. Opatrna 22. Picea abies somatic embryo development from suspension cultures and agar-based cultures: a comparison Christopher Hunter & Lionel Levy 23. Somatic embryogenesis by liquid culture of epidermal layers in sunflower: from genetic control to cell development Michel Petitprez, A. Sarrafi, E. Flores-Berrios, X. Xuhan, C. Briere & L. Gentzbittel 24. Development of photoautotrophy in Coffea somatic embryos enables mass production of clonal transplants F.

Embryo-specific Proteins in Cyclamen persicum Analyzed with 2-D DIGE

Somatic embryogenesis can be used to produce artificial seeds of Cyclamen persicum, one of the most important ornamental plants for the European market, both as a potted plant in northern Europe and a bedding plant in the cool winters in southern Europe. The aim of this study was to obtain new insights into the molecular biology of somatic embryogenesis, which in turn can be useful for the improvement of tissue culture methodology. Total proteins were characterized from two isogenic cell lines of Cyclamen persicum, one that was embryogenic and one that never has shown any embryogenic capacity. The extracted proteins were separated by two-dimensional differential gel electrophoresis (2-D DIGE) and selected proteins were treated using the ETTAN Dalt Spot Handling Workstation. Protein identification was performed using MALDI-TOF-MS. More than 1200 Cyclamen proteins were detected; 943 proteins were common to both lines. The different protein patterns of the embryogenic and non-embryogenic cell lines were obvious: One hundred eight proteins were more abundant in the embryogenic cells, and 97 proteins in the non-embryogenic cells. Among the differentially expressed proteins, 128 were identified. MALDI-TOF-MS analysis enabled 27 spots to be proposed as candidates for embryo-specific proteins, as they were unique to the embryogenic cell line. The proteins identified are involved in a variety of cellular processes, including cell proliferation, protein processing, signal transduction, stress response, metabolism, and energy state, but the majority are involved in protein processing and metabolism. The main functions of the putative embryo-specific proteins have been discussed in proportion to their role in the somatic embryogenesis process.

Sequential cell wall transformations in response to the induction of a pedicel abscission event in Euphorbia pulcherrima (poinsettia).

Alterations in the detection of cell wall polysaccharides during an induced abscission event in the pedicel of Euphorbia pulcherrima (poinsettia) have been determined using monoclonal antibodies and Fourier transform infrared (FT-IR) microspectroscopy. Concurrent with the appearance of a morphologically distinct abscission zone (AZ) on day 5 after induction, a reduction in the detection of the LM5 (1-->4)-beta-D-galactan and LM6 (1-->5)-alpha-L-arabinan epitopes in AZ cell walls was observed. Prior to AZ activation, a loss of the (1-->4)-beta-D-galactan and (1-->5)-alpha-L-arabinan epitopes was detected in cell walls distal to the AZ, i.e. in the to-be-shed organ. The earliest detected change, on day 2 after induction, was a specific loss of the LM5 (1-->4)-beta-D-galactan epitope from epidermal cells distal to the region where the AZ would form. Such alteration in the cell walls was an early, pre-AZ activation event. An AZ-associated de-esterification of homogalacturonan (HG) was detected in the AZ and distal area on day 7 after induction. The FT-IR analysis indicated that lignin and xylan were abundant in the AZ and that lower levels of cellulose, arabinose and pectin were present. Xylan and xyloglucan epitopes were detected in the cell walls of both the AZ and also the primary cell walls of the distal region at a late stage of the abscission process, on day 7 after induction. These observations indicate that the induction of an abscission event results in a temporal sequence of cell wall modifications involving the spatially regulated loss, appearance and/or remodelling of distinct sets of cell wall polymers.

Engineering aspects of plant propagation in bioreactors

Millions of plants are produced every year through micropropagation worldwide. However, these methods are labour intensive and there has to be a good reason for choosing micropropagation. Either the crop is so valuable in itself, or the product from micropropagation is of superior quality and therefore able to obtain a higher price. Or, traditional propagation is so costly, difficult or impossible that micropropagation solves a problem (Hvoslef-Eide 1987; Aitken-Christie 1991). Jones and Sluis (1991) claim that price is the single greatest barrier to true exploitation of mass production opportunities. Automation of somatic embryogenesis/organogenesis in bioreactors has been advanced by several authors, among them Styer (1985), Preil et al. (1988) and Levin et al. (1988), as a possible way of reducing the labour costs of micropropagation. Bioreactors have traditionally been used for bacterial fermentation or for large scale production of secondary metabolites from plant cells. Growing plants cells for production of somatic embryos in bioreactors designed for bacterial growth and production of secondary metabolites is not as straightforward as first thought. Experience from a European collaboration on regeneration from suspension cultures has lead us to believe that the shear forces from fast propellers and foam formation in bubble aerated reactors seem to have caused most trouble.

Bioreactor design for propagation of somatic embryos

AbstractSix identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (‘clonal or somatic seeds’). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a slow-speed stirrer that regularly changes direction of rotation to prevent ‘quiet’ zones in the suspension in which cells can settle and grow. In addition, the oxygen is provided, bubble-free, through thin silicone tubing loops that are hanging loose, moving with the liquid to prevent cell growth on these tubes. We used off-the-shelf components whenever possible, to reduce the costs to a minimum, which was another aim of the construction. The result was a suite of relatively inexpensive computer-controlled bioreactors that could control temperature, oxygen, pH, stirrer speed and stirrer direction. In addition, we have provided different light spectral qualities by simple means of filtering the light. Using the present software, the parameters can be set up to alter every hour during the 24-h day/night cycle. All our cultures have improved growth in the bioreactors compared to identical cultures in Erlenmeyer flasks. The cultures used are: embryogenic cultures of carrot (Daucus carota), Norway spruce (Picea abies), birch (Betula pendula), cyclamen (Cyclamen persicum) and shoot cultures of Christmas begonia (Begonia x cheimantha). The paper also discusses recommendations for improvements of the current system for future revisions.

Characterization and structural analysis of wild type and a non-abscission mutant at the development funiculus (Def) locus in Pisum sativum L

BackgroundIn pea seeds (Pisum sativum L.), the Def locus defines an abscission event where the seed separates from the funicle through the intervening hilum region at maturity. A spontaneous mutation at this locus results in the seed failing to abscise from the funicle as occurs in wild type peas. In this work, structural differences between wild type peas that developed a distinct abscission zone (AZ) between the funicle and the seed coat and non-abscission def mutant were characterized.ResultsA clear abscission event was observed in wild type pea seeds that were associated with a distinct double palisade layers at the junction between the seed coat and funicle. Generally, mature seeds fully developed an AZ, which was not present in young wild type seeds. The AZ was formed exactly below the counter palisade layer. In contrast, the palisade layers at the junction of the seed coat and funicle were completely absent in the def mutant pea seeds and the cells in this region were seen to be extensions of surrounding parenchymatous cells.ConclusionThe Def wild type developed a distinct AZ associated with palisade layer and counterpalisade layer at the junction of the seed coat and funicle while the def mutant pea seed showed non-abscission and an absence of the double palisade layers in the same region. We conclude that the presence of the double palisade layer in the hilum of the wild type pea seeds plays an important structural role in AZ formation by delimiting the specific region between the seed coat and the funicle and may play a structural role in the AZ formation and subsequent detachment of the seed from the funicle.

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 μmol m−2 s−1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 μmol m−2 s−1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 μmol m-2 s-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 μmol m-2 s-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 μmol m-2 s-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.

Growth, seed development and genetic analysis in wild type and Def mutant of Pisum sativum L

BackgroundThe def mutant pea (Pisum sativum L) showed non-abscission of seeds from the funicule. Here we present data on seed development and growth pattern and their relationship in predicting this particular trait in wild type and mutant lines as well as the inheritance pattern of the def allele in F2 and F3 populations.FindingsPod length and seed fresh weight increase with fruit maturity and this may affect the abscission event in pea seeds. However, the seed position in either the distal and proximal ends of the pod did not show any difference. The growth factors of seed fresh weight (FW), width of funicles (WFN), seed width (SW) and seed height (SH) were highly correlated and their relationships were determined in both wild type and def mutant peas. The coefficient of determination R2 values for the relationship between WFN and FW, SW and SH and their various interactions were higher for the def dwarf type. Stepwise multiple regression analysis showed that variation of WFN was associated with SH and SW. Pearsons chi square analysis revealed that the inheritance and segregation of the Def locus in 3:1 ratio was significant in two F2 populations. Structural analysis of the F3 population was used to confirm the inheritance status of the Def locus in F2 heterozygote plants.ConclusionsThis study investigated the inheritance of the presence or absence of the Def allele, controlling the presence of an abscission zone (AZ) or an abscission-less zone (ALZ) forming in wild type and mutant lines respectively. The single major gene (Def) controlling this phenotype was monogenic and def mutants were characterized and controlled by the homozygous recessive def allele that showed no palisade layers in the hilum region of the seed coat.

Primary and Secondary Abscission in Pisum sativum and Euphorbia pulcherrima—How Do They Compare and How Do They Differ?

Abscission is a highly regulated and coordinated developmental process in plants. It is important to understand the processes leading up to the event, in order to better control abscission in crop plants. This has the potential to reduce yield losses in the field and increase the ornamental value of flowers and potted plants. A reliable method of abscission induction in poinsettia (Euphorbia pulcherrima) flowers has been established to study the process in a comprehensive manner. By correctly decapitating buds of the third order, abscission can be induced in 1 week. AFLP differential display (DD) was used to search for genes regulating abscission. Through validation using qRT-PCR, more information of the genes involved during induced secondary abscission have been obtained. A study using two pea (Pisum sativum) mutants in the def (Developmental funiculus) gene, which was compared with wild type peas (tall and dwarf in both cases) was performed. The def mutant results in a deformed, abscission-less zone instead of normal primary abscission at the funiculus. RNA in situ hybridization studies using gene sequences from the poinsettia differential display, resulted in six genes differentially expressed for abscission specific genes in both poinsettia and pea. Two of these genes are associated with gene up- or down-regulation during the first 2 days after decapitation in poinsettia. Present and previous results in poinsettia (biochemically and gene expressions), enables a more detailed division of the secondary abscission phases in poinsettia than what has previously been described from primary abscission in Arabidopsis. This study compares the inducible secondary abscission in poinsettia and the non-abscising mutants/wild types in pea demonstrating primary abscission zones. The results may have wide implications on the understanding of abscission, since pea and poinsettia have been separated for 94–98 million years in evolution, hence any genes or processes in common are bound to be widespread in the plant kingdom.

Macro- and micronutrient nutrition of plants in greenhouses, hydroponic systems, and in vitro culture on gelled media

Nutrition for in vitro and greenhouse production systems is reviewed and found to be broadly similar. The optimal pH (5.0–6.0) is independent of the growing system used, with some lower or higher pH values required for special crops. The pH of nutrient solutions is regulated either with acid or via the NH4:NO3 ratio. For in vitro solutions, it is possible to use buffers (MES or TRIS), while in greenhouse systems the pH buffering capacity relies mostly on the quantity of colloids in the growing medium.