Micropropagation of Selected Agave Species

Micropropagation of selected Agave species

Dariusz KULUS∗

Keywords: CAM; in vitro regeneration; plant tissue culture Abstract: Agaves are a very important group of plants. They are popular ornamentals but they are also used in the production of drugs, cosmetics, drinks, food and fodder. Unfortunately, due to the growing inﬂuence of anthropopressure, some of them are threaten with extinction. Therefore, in order to always be able to meet the growing demands of the market, novel biotechnological tools need to be applied in the production of these species. Micropropagation, i.e. vegetative multiplication of plants under aseptic, strictly controlled conditions and with the use of synthetic media, is the most commonly applied aspect of plant tissue cultures. The technique reduces time, space and costs required for the production of plants. Over time, several micropropagation techniques have been developed also with agaves. The aim of the present review is to present the current achievements and problems associated with micropropagation of the most important agave species.

1. Introduction: origin and uses

The genus Agave contains 155 species (and over 200 varieties) of the Agavaceae family, 75 % of which are native to Mexico. They are found from South America northwards to Mexico, and beyond to the southern States of America, as well as up to the coast of California, and in the Caribbean Islands. The genus was established by Linnaeus in 1753 (Debnath et al., 2010). Agave has been a renewable source for food, beverages (tequila), ﬁbers (sisal), silage for livestock, drugs (saponins, sterols, steroidal alkaloids, alkaloidalamines), ornamental plants (due to their distinctive leaf form and color) and other useful products. Some of the common agaves are: Agave aboriginum, Agave abortiva, Agave abrupta, Agaveacklinicola, Agave aﬃnis, Agave albescens, Agave al- bomarginata, Agave alibertii, Agave aloides, Agave amaniensis, Agave americana, Agave angustifolia, Agave angustissima, Agave anomala, Agave antillarum, Agave arizonica, Agave arubensis, Agave aspera, Agave asperrima, Agave atrovirens, Agave atrovirens var. mirabilis, Agave attenuata, avellanidens, Agave bovicornuta, Agave bracteosa, Agave brauniana, Agave breedlovei, Agave brevipetala, Agave breviscapa, Agave brevispina, Agave brittonia, Agave bromeliaefolia, Agave brunnea, Agave bulb-

∗University of Technology and Life Sciences in Bydgoszcz, Faculty of Agriculture and Biotechnol- ogy, Department of Ornamental Plants and Vegetable crops – Laboratory of Biotechnology 76 PhD Interdisciplinary Journal ifera, Agave cantala, Agave caymanensis, Agave chiapensis, Agave corderoyi, Agave costaricana, Agave cucullata, Agave cundinamarcensis, Agave cupreata, Agave de- serti, Agave donnell-smithii, Agave durangensis, Agave dussiana, Agave eggersiana, Agave falcata, Agave filifera, Agave fourcroydes, Agave geminiflora, Agave acicularis, Agave aurea, Agave Agaveimpressa, Agave jaiboli, Agave lechuguilla, Agave lophantha, Agave macroculmis, Agave mckelveyana, Agave neglecta, Agave palmeri, Agave parv- iflora, Agave ragusae, Agave rasconensis, Agave regia, Agave revoluta, Agave rhoda- cantha, Agave rigida, Agave roezliana, Agave rudis, Agave rupicola, Agave salmiana, Agave schottii, Agave scolymus, Agave simoni, Agave sisalana, Agave stricta, Agave stringens, subinermis, Agave subsimplex, Agave subtilis, Agave subzonata, Agave sul- livani, Agave tequilana, Agave toumeyana, Agave tubulata, Agave utahensis, Agave victoriae-reginae, Agave vivipara, Agave weberi, Agave xylonacantha, Agave yuccae- folia, Agave zebra (Debnath et al., 2010). Agave tequilana Weber cultivar azul is the most widely cultivated Agave species. Its plantations encompass more than 84.000 ha along five Mexican states, representing millions of dollars of income (Santacruz- Ruvalcaba and Portillo, 2009). At the same time over-collection, habitat destruction and a long-life span have significantly reduced populations of some species in their native habitat (Kański, 2003). Therefore, developing efficient multiplication protocols is justified.

2. Reproduction of Agaves

Plants can be propagated generatively through seeds or vegetatively, e.g. by stolon cuttings, bulbils produced in the inflorescence, and grafting (Lema-Rumińska and Kulus, 2014). Generative reproduction is often highly efficient, since plants produce numerous seeds. However, some succulents (e.g. of the Agave genera) are hapaxan- thic, i.e. they flower only once in their lifetime (as for Agave species it may take even 10 – 90 years) and die subsequently. Others, of hybrid origin (e.g. Agave sisalana) are sexually sterile and unable to produce seeds (Debnath et al., 2010). Moreover, the method does not guarantee genetic stability, since seeds are produced as a conse- quence of a fusion of gametes belonging to two different organisms. This is a problem for plants of unique morphology. For example, there was only one individual of Astro- phytumasterias (Zucc.) Lem. ’Superkabuto’– a very popular collectors plant, found in nature. Also the black-epidermis Copiapoatenuissima F. Ritter, and white-spine Gymnocalyciumgibbosum (Haw.) Pfeiff. ex Mittler do not maintain their features when propagated generatively. Clonal reproduction is possible after applying vegetative multiplication methods. These techniques, on the other hand, are less efficient (for example, grafting of 1 ha Gymnocalyciummichanovichii Britton & Rose continues for two years) and is threatened with transferring virus diseases (Jeong et al., 2004). Moreover, traditional cultivation in the field or in the glasshouse requires large areas and financial investment. At the same time such collections are threatened with both biotic (disease, pests and insects) and abiotic stress. Succulents are very susceptible to rots caused by bacteria and fungi (Gratton and Fay, 1999). Therefore, today, in order to satisfy the demands of the market, novel biotechnological tools need to be applied. Among them, in vitro tissue cultures are the most basic ones. Plant biotechnology utilizes a range of in vitro techniques to manipulate germplasm, including: clonal reproduction, generation of novel genotypes, metabo- 77 lites acquisition, and storage/protection of valuable genetic resources (Al-Ramamneh et al., 2006a,b; Lema-Rumińska and Kulus, 2012), Fig.1. They are also used for do- mestication of plants when traditional methods fail, as it was done with Yuccavalida Brandegee – a species valuable for the production of steroid saponins (Szopa and Kośtyń, 2006). At the same time they are used to introduce new, unknown species onto the market.

Fig. 1. Various aspects of plant tissue culture utilization.

Nowadays in vitro tissue culture-based micropropagation is considered to be the most efficient plant reproduction method. In this technology small pieces of plants called explants are cultured on synthetic nutrient media, in aseptic, strictly controlled light and temperature conditions. Micropropagation combines the advantages of the previously described methods: yield and stability. Furthermore in vitro-produced plants are often characterized by a quicker growth-pace and a better condition, as observed with Kalanchoetubiflora (Harvey) Hamet. For example, some succulents grow 1 cm even 4 years in nature and 3 years in the glasshouse, while under in vitro conditions their growth is even three to seven times accelerated (Pérez-Molphe-Balch and Dávila-Figueroa,2002). Therefore, the use of the method is growing annually. Every year about two billiards of plants from 160 genera are produced on the basis of micropropagation (Rout et al., 2006). Most of them (80 – 90 %) are ornamental plants. Micropropagation techniques found wide applications with several succulents of the Agave (Robert et al., 1987), Aloes (Choudhary et al., 1987), Hylotelephium (Nakano et al., 2005), Sedum (Wojciechowicz, 2009), and Kalanchoe (Saifullah et al., 2006) genera. Each year this list is supplemented with new species. The aim of this study is to present previous and current achievements with the 78 PhD Interdisciplinary Journal micropropagation of selected agave species.

3. Micropropagation of Agave

Agaves belong to a specific group of CAM plants (Crassulacean Acid Metabolism, succulents – nocturnal acid accumulation). This specificity is also reflected in terms of in vitro cultures requirements. Over time several agave micropropagation techniques have been developed (Kulus, 2014), Fig. 2. Much of the tissue culture work in agaves has been carried out on ornamental species and focused on improving multiplication rates (Debnath et al., 2010). Selection of the optimal protocol depends on the genotype, the level of technical skill, and on the objective one wants to achieve. Usually for micropropagation a popular MS (Murashige and Skoog, 1962) medium is used with pH 5.6 – 5.8 and solidified with 0.7 – 0.9 % agar. As for agave, micropropagation in liquid media has not yet been reported as a successful tool, even though it helps to automate the micropropagation process (Santacruz-Ruvalcaba and Portillo, 2009).

Fig. 2. Integration of various plant tissue culture types (Walden and Wingender, 1995).

3.1. Explant preparation and disinfection Before explants excision any dead, damaged or rotting tissues should be removed from the mother plant. If the plant is infected with pests, then it should be dipped in 95 % ethanol for 10 – 30 s and then wiped with a soft tissue. Any adhering soil should be removed by washing under sterile water containing a few drops of Tween 80. Woody spines and hairs are difficult to surface-disinfect effectively, so they can be removed, using forceps, needles or scalpels. After excision from the mother plant, the explants need to be surface disinfected. The fragments of plant tissue should be left as large as possible for disinfection, to minimize damage to tissues by the disinfectant (Gratton and Fay, 1999). There are many disinfection agents available such as: sodium or calcium hypochlorite, mercuric 79 chloride. Among them, sodium hypochlorite (0.3 – 14 %) is the most frequently used because it is easily water-soluble. Disinfection time ranges from 5 to 15 minutes. Surface disinfection can be improved if the vessel is put on a magnetic stirrer set at a speed high enough to keep the biological material submerged, but not too high to prevent tissue damage (Gratton and Fay, 1999). Afterwards the explants should be rinsed three times with distilled, sterile water to stop the activity of the disinfectant. 3.2. Axillary buds activation and rhizogenesis Members of the Agavaceae family are often propagated through axillary shoots proliferation. The first reports on this method were ineffective with Agave sisalana (Das, 1992), however, later research performed with Agave parrasana was much more promising (Santacruz-Ruvalcaba et al., 1999). In this technique, axillary buds present in the leaves axils get activated as a result of cytokinin’s presence in the culture medium (preferably BA – benzylade- nine, but also KIN – kinetin or TDZ – thidiazuron). To stimulate the elongation of shoots, auxins (e.g. NAA - 1-naphthaleneacetic acid) are also added. However, 2,4-D (2,4-dichlorophenoxiacetic acid) drastically inhibits shoot proliferation (Santacruz- Ruvalcaba et al., 1999). Addition of 0.4 µM NAA + 0.5 µM indole-3-butyric acid (IBA) + 2.3 µM KIN caused an extensive proliferation of multiple shoot primordial form stolones of Agave cantala Roxb., A. fourcroydes Lem. and A. sisalana (Binh et al., 1990). Rooting occurs spontaneously or in the presence of IBA. Interestingly, as for Agave parrasana Berger it was observed that higher light intensity (100 µM·m−2 · s−1) stimulated better rhizogenesis than standard 25 100 µM·m−2·s−1 (Santacruz-Ruvalcaba et al., 1999). 3.3. Caullogenesis Proliferation through axillary shoots is a genetic stability-guarantee (due to the presence of meristematic tissue); however, its yield is limited due to the necessity of using a meristematic explants (Kulus, 2014). Adventitious organogenesis is much more efficient, since it can be used to produce shoots from practically any explant (internode, petal, root segment, leaf, etc.) Callus was regenerated from in vitro-grown immature leaves, ex vitro-grown ma- ture leaves and rhizome explants of Agave sisalana on MS medium + 9.05 µM 2,4-D + 4.6 µM KIN or 9.05 µM 2,4-D + 4.6 µM KIN or modified MS (NH4NO3, 1500 mg·dm−3) + 9.05 µM 2,4-D + 4.6 µM KIN (Hazra et al., 2002). Light was essential for callus formation. Shoot regeneration from callus was only achieved from rhizomes and immature leaves. The highest regeneration rate was obtained with BA (26.6 µM) as the only PGR (plant growth regulator). Shoot proliferation rate increased on half-strength (1/2 MS) medium supplemented with BA (8.9 µM). Microshoots were rooted on MS medium with IAA (11.42 µM). Acclimatization and transfer to soil was 100 % successful. Callus of Agave sisalana was also initiated from rhizome, and stem explants on MS, SH, Gamborgand White’s medium containing various concentrations of BA, kinetin, NAA, IAA and 2,4-D either in combination or solely. The highest numbers of shoots were produced from stem and rhizome explants directly or from callus. The capacity 80 PhD Interdisciplinary Journal for shoot regeneration remained constant in the callus for more than 32 months. A total of 100 % of the rooted plants were successfully adapted to field conditions and grown in the soil. Regenerated plants were morphologically similar to the field grown mother plants (Nikam, 1997).

3.4. Somatic embryogenesis

Somatic embryogenesis is a process during which non-zygotic plant cells, including haploid cells, form embryos, and ultimately, fertile plants (Rose et al., 2010). Since plants poses millions of somatic cell, which may acquire competence to perform embryogenesis, therefore, the yield of the technique is enormous. Today somatic embryogenesis is believed to be the most efficient micropropagation method (Kulus, 2013). Somatic embryos are bipolar structures (with both apical and root poles). There- fore, in contrast to axillary and adventitious shoots, they do not require additional transfer to a rooting medium. Moreover, somatic embryos of Agave tequilana have a unicellular origin, so there is no risk of forming chimeras (Portillo et al., 2007; Santacruz-Ruvalcaba and Portillo, 2009). As for other Agave species, somatic embryos have a multicellular origin, which may lead to genetic mutation, especially if regeneration is indirect, through callus (Martinez-Palacios et al., 2003). With Agavesisalana Perr. Ex. Engelm most of the embryos had a distinct multicellular origin as they developed from epidermal, sub-epidermal and inside callus cells. Only a few of them originated from a superficial callus cell (Nikam et al., 2003). Regeneration (induction) of somatic embryos is usually stimulated by the presence of 2,4-D auxin (2.3 – 9.0 µM) in the culture medium. However, the presence of cytokinins (especially BA) may also be beneficial. With Agave tequilana it was even observed, that embryos regenerated on the medium with high cytokinin concentration were green, in contrast to those from the medium with higher concentration of auxins (Portillo et al., 2007). This phenomenon is caused by the fact that cytokinins stimulate chlorophyll synthesis. Green embryos are more preferable since they show a greater chance of conversion into plantlets. In general the ratio of auxin/cytokinin has to be optimized individually for every species. Unlike other species, an increase in sucrose concentration (higher than the standard 3 %) does not increase the embryogenesis efficiency of agave (Santacruz-Ruvalcaba and Portillo, 2009)).To stimulate further development of somatic embryos, they often need to be transferred on a auxin-free medium (Nikam et al., 2003). Portillo et al. (2007) observed also that regeneration of somatic embryos is strongly influenced by genotype (0.83 – 394).

3.5. Thin cell suspension layer

This hybrid technology fuses the advantages, ﬁrstly, of a liquid culture made through cellular dissociation to form a cellular suspension of embryonic callus, and secondly the convenience of a solid medium for somatic embryogenesis expression and development (Santacruz-Ruvalcaba and Portillo, 2009). With Agave tequilana TCL encouraged complete expression of embryoids without transfer to extra media cultures, and a higher number of generated embryos was obtained (Santacruz-Ruvalcaba and Portillo, 2009). 81

Tab. 1. Micropropagation protocols and eﬃciencies of selected succulent species. L2 vitamin – (Phillips and Collins, 1999), SH – (Schenk and Hildebrandt, 1972).

Species Agaveparrasana Agave victoria- Agave sisalana reginae Explant Seed oﬀshoots Seedling stem Young leaves Young shoots fragments Protocol MS + L2 vita- MS/SH + L2 MS + 0.88-44 MS + 0.9 µM mins + 53.2 µM vitamins + 2.2 µM BA 2,4-D + 4.44 BA µM BA µM BA then transferred to MS + 0.46 µM KIN Eﬃciency 48.6 2.1 0 6.24 shoots/ explant Reference (Santacruz- (Martinez- (Das, 1992) (Nikam et al., Ruvalcaba Palacios et al., 2003) et al., 1999) 2003)

4. Problems of culture establishment

Even though micropropagation has numerous advantages, there are also some obsta- cles, which need to be considered. The main problems associated with plant tissue cultures are:

• Some species or tissues have low regeneration potential.

• A serious and, unfortunately, still common issue associated with somatic embryos is their asynchronous development, and the problem with conversion into microshoots caused by an abnormal apical meristem development. As for Agave sisalana a maximum 75.7 % germination and 65.7 % plantlet formation was observed after transferring the produced embryos on a kinetin-supplemented medium (Nikam et al., 2003).

• Problems with explants disinfection (presence of endophytes) and human errors during tissue transfers which may result in contamination development and loss of the material.

• Possible occurrence of somaclonal variation.

• Sometimes, due to high humidity in the culture vessel, improper gelling agent concentration, pH or presence of some PGRs shoots are hyperhydrated (the so called vitriﬁcation) and show a glassy appearance. With Agave tequilana, this 82 PhD Interdisciplinary Journal

phenomenon persists even with the use of a temporary immersion system (TIS). TCL is proposed to solve this problem (Santacruz-Ruvalcaba and Portillo, 2009). • Since in vitro conditions are much diﬀerent from the natural ones, some plants show problems with acclimatization to ex vitro conditions. Succulents, however, possess a thick cuticle and are often characterized by a compact body shape, therefore, they are free from the desiccation shock frequently observed with other plants (Lema-Rumińska and Kulus, 2014). As for Agave victoria-reginae, 90 % of somatic embryos and 94 % of axillary shoots survived acclimatization and grew into healthy plants (Martinez-Palacios et al., 2003). • The main limiting factor is high labor costs (reaching even 60 – 70 % of the total production costs). These expenditures can be reduced through procedure automation (e.g. application of bioreactors).

5. Conclusion

There are a number of factors, which can significantly influence the efficiency of agave micropropagation, which should be considered when optimizing the culture conditions; these include among others: • the genotype and condition (health and vigor) of the source plant; • the explants tissue type and the way of its inoculation on the medium; • the physiological age and size of the explants; • the season in which the in vitro culture was initiated; • PGRs (type and concentration) used; • gelling agent and carbon/nitrogen source; • physical conditions in the growth room (mainly light conditions). Still, it is usually possible to find such an explants source and optimize culture conditions to achieve satisfying results. Therefore, it can be assumed that laboratory reproduction is the future of agave production. References Al-Ramamneh, E., S. Sriskandarajah and M. Serek (2006b), ‘Plant regeneration via somatic embryogenesis in schlumbergera truncata’, Plant Cell, Tissue and Organ Culture 84 pp. 333–342. Al-Ramamneh, E.A., S. Sriskandarajah and M. Serek (2006a), ‘Agrobacterium tumefaciens-mediated transformation of rhipsalidopsis gaertneri’, Plant Cell Re- ports 25(11) pp. 1219–1225. Binh, L.T., L.T. Muoi, H.T.K. Oanh, T.D. Thang and D. T. Phong (1990), ‘Rapid propagation of agave by in vitro tissue culture’, Plant Cell, Tissue and Organ Culture 23(1) pp. 67–70. Choudhary, A. K., A. K. Ray, S. Jha and I. N. Mishra (1987), ‘Callus formation, shoot initiation and in vitro culture of aloe vera’, Bioinformatics and Bioengineering 1(4) pp. 551–553. 83

Das, T. (1992), ‘Micropropagation of agave sisalana’, Plant, Cell, Tissue and Organ Culture 31(3) pp. 253–255. Debnath, M., M. Pandey, R. Sharma, G. S. Thakur, P. Lal, M. Debnath and M. Pandey (2010), ‘Biotechnological intervention of agave sisalana: a unique fiber yielding plant with medicinal property’, Journal of Medicinal Plants Research 4(3) pp. 177–187. Gratton, J. and F.M. Fay (1999), ‘In vitro propagation of succulent plants’, Plant Cell Culture Protocols – Methods in Molecular Biology 111 pp. 135–140. Hazra, S-K., S. Das and A-K. Das (2002), ‘Sisal plant regeneration via organogenesis’, Plant Cell, Tissue and Organ Culture 70(3) pp. 235–240. Jeong, I.M., H.C. Cho and J.M. Lee (2004), ‘Production and breeding of cacti for grafting in korea’, Chronica Horticulturae 44(3) pp. 7–10. Kański, J. (2003), ‘The succulent flora of mexico - the threatments and conservation’, Biuletyn Ogrodów Botanicznych 12 pp. 187–189. Kulus, D. (2013), Embriogeneza somatyczna in vitro, CreativeTime, Kraków. Kulus, D. (2014), Biotechnological methods of ornamental plants reproduction, Insty- tut Naukowo-Wydawniczy Maiuscula. Lema-Rumińska, J. and D. Kulus (2012), ‘Induction of somatic embryogenesis in astrophytum asterias (zucc.) lem. in the aspect of light conditions and auxin 2,4-d concentrations’, Acta Scientiarum Polonorum, Hortorum Cultus 11(4) pp. 77–87. Lema-Rumińska, J. and D. Kulus (2014), ‘Micropropagation of cacti – a review’, Haseltonia 19 pp. 46–63. Martinez-Palacios, A., M. Ortega-Larroccea and V.M. Chavez (2003), ‘Somatic embryogenesis and organogenesis of agave victoriae-reginae: considerations for its conservation’, Plant Cell, Tissue and Organ Culture 74 pp. 135–142. Murashige, T. and F. Skoog (1962), ‘A revised medium for rapid growth and bio assays with tobacco tissue cultures’, Physiologia Plantarum 15 pp. 473–497. Nakano, M., M. Nagai, S. Tanaka, M. Nakata and T. Godo (2005), ‘Adventitious shoot regeneration and micropropagation of the japanese endangered hylotelephium sieboldii (sweet ex hook.) h. ohba and h. sieboldii var. ettyuense (tomida)’, Plant Biotechnology 22(3) pp. 221–224. Nikam, T.D. (1997), ‘High frequency shoot regeneration in agave sisalana’, Plant Cell, Tissue and Organ Culture 51(3) pp. 225–228. Nikam, T.D., G.M. Bansude and K.C. Kumar (2003), ‘Somatic embryogenesis in sisal (agave sisalana perr. ex. engelm)’, Plant Cell Report 252 pp. 188–194. Phillips, G.C. and G.B. Collins (1999), ‘In vitro tissue culture of selected legumes and plant regeneration from callus culturesof red clover’, Crop Sciences 19 pp. 59–64. Portillo, L., R. Santacruz-Ruvaleaba, A. Gutierez-Mora and B. Rodriguez-Garay (2007), ‘Somatic embryogenesis in agave tequilana weber cultivar azul’, In vitro Cellular and Developmental Biology – Plant 43 pp. 569–575. Pérez-Molphe-Balch, E. and C. Dávila-Figueroa(2002), ‘In vitro propagation of pele- cyphora aselliformis ehrenberg and p. strobiliformis werdermann (cactaceae)’, In Vitro Cellular and Development Biology - Plant 38(1) pp. 73–78. Robert, M. L., J. L. Herrera, F. Contreras and K. N. Scorer (1987), ‘In vitro propagation of agave fourcroydes lem. (henequen)’, Plant Cell, Tissue and Organ Culture 8 pp. 37–48. Rose, R.J., F.R. Mantiri, S. Kordyukov, S-K. Chen, X-D. Wang, K.E. Nolan and M.B. Sheahan (2010), Development biology of somatic embryogenesis, Springer-Verlag, Berlin. Rout, G. R., A. S. Mohapatra and J. Mohan (2006), ‘Tissue culture of ornamental pot plant: a critical review on present scenario and future prospects’, Biotechnology Advances 24(6) pp. 531–560. Saifullah, K., N. Sheeba, A. Kashif and Z. Samreen (2006), ‘Direct organogenesis of kalanchoe tomentosa (crassulaceae) from shoot-tips’, Plant Tissue Culture and Biotechnology Division 38(4) pp. 981–997. 84 PhD Interdisciplinary Journal

Santacruz-Ruvalcaba, F., H. Gutierrez-Pulido and B. Rodrigez-Garay (1999), ‘Eﬃ- cient in vitro propagation of agave parrasana berger’, Plant Cell, Tissue and Organ Culture 56 pp. 163–167. Santacruz-Ruvalcaba, F. and L. Portillo (2009), ‘Thin cell suspension layer as a new methodology for somatic embryogenesis in agave tequilana weber cultivar azul’, Industrial Crops and Products 29(2-3) pp. 609–614. Schenk, R.U. and A.C. Hildebrandt (1972), ‘Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures’, Canadian Journal of Botany 50 pp. 199–204. Szopa, J. and K. Kośtyń (2006), ‘Kultury komórkowe i rośliny transgeniczne w biotechnologii’, Biotechnologia 4(75) pp. 7–17. Walden, R. and R. Wingender (1995), ‘Gene-transfer and plant-regeneration techniques’, Trends in Biotechnology 13 pp. 324–331. Wojciechowicz, M. K. (2009), ‘Organogenesis and somatic embryogenesis induced in petal cultures of sedum species’, Acta Biologica Cracoviensia Series Botanica 51(1) pp. 83–90.