In Vitro Micropropagation of Calla Lily: an Overview

Kumar and Dogra Available Ind. J. Pure online App. Biosci. at www.ijpab.com (2020) 8(2), 144 -153 ISSN: 2582 – 2845

DOI: http://dx.doi.org/10.18782/2582-2845.7974 ISSN: 2582 – 2845 Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 Review Article

In vitro Micropropagation of Calla lily: An Overview

Akhil Kumar1* and Ishani Dogra2 1Department of Biotechnology, Dr. Y S Parmar University of Horticulture and Forestry Nauni, Solan (H.P)-173230, India 2Department of Biotechnology, Swami Keshwanand Agriculture University Bikaner, Rajasthan, India *Corresponding Author E-mail: [email protected] Received: 2.03.2020 | Revised: 7.04.2020 | Accepted: 11.04.2020

ABSTRACT Calla lily belonging to the Araceae family is used as a cut flower and in the landscaping of gardens. Plant parts such as leaves, rhizomes, roots, stems, and the whole plant are used as herbal medicine. Conventional methods used for propagation of calla lily do not produce adequate planting material as there is various fungal, bacterial and virus attack on the plant during plant propagation which leads to poor yield in flower and rhizome or some time it causes the death of the plant. Therefore, plant tissue culture is the only technique that allows fast clonal propagation also provides healthy and uniforms plants. In most cases, growth regulators like BAP, IBA, and NAA were found to be essential for growth, multiplication of shoot and root formation. Some species of the calla lily are also used as herbal medicine having activity such as antibacterial, antifungal, antioxidant, antihistaminic, antialgal, antithrombotic, and anticoagulant activities. Therefore results show that there is a need for improvement in further research and development that need to be carried out to improve this crop, to developing improved varieties with commercially significant yield. In the present review, micropropagation methods and protocols are combined and gather the information to researchers for further investigation and to produce quality as well as quantity planting material.

Keywords: Rhizome, Antifungal, Anticoagulant, Plant tissue culture, BAP

INTRODUCTION coloured calla has several different colours Calla lily, Zantedeschia spp. ‘Southern Light’ such as yellow, red, and purple which makes it is a bulbous plant belonging to the as a cut flower, a potted flower, and a garden genus Zantedeschia. It is native to Southern plant. In the early 1990s, the calla industry Africa and mainly distributed at an altitude grew rapidly, with New Zealand's exports of range from 1,000 to 2,000 meters in temperate cut flower increasing by 75% in five years, regions of South Africa, Zimbabwe, and and an effort made to produce bulbs Nigeria (Letty, 1973). It has been widely used throughout the year (Clemens & Eddie Welsh, as an ornamental plant because it has a unique 1993). leaf shape like flowers. Particularly, the

Cite this article: Kumar, A., & Dogra, I. (2020). In vitro Micropropagation of Calla lily: An Overview, Ind.

J. Pure App. Biosci. 8(2), 144-153. doi: http://dx.doi.org/10.18782/2582-2845.7974

Kumar and Dogra Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 ISSN: 2582 – 2845 Small rhizomes that are overwintered in pots the induction dedifferentiation in Zantedeschia under cover can be cut up into sections, and spreng, cytokinins are commonly used for each of them has a visible bud. Large callus formation (D’Arth et al., 2002; Naor et overwintered clumps in the garden can be split al., 2004; Yip et al., 2006). Indirect by lifting the plant before there is much top organogenesis offers strategy for growth, and cutting through the roots with a dedifferentiating plant tissue into callus spade and isolating into smaller sections. permitting transformation, selection Nevertheless, large-scale production of the procedures, and regenerating processes for calla lily plants is narrow by tuber development of in vitro whole plants from propagation, which is the traditional transformed callus (Robinson and Firozabad, reproduction method used which result in low 1993). A variety of type of explants, such as propagation coefficient. Specifically, because rhizomes and buds (Chang et al., 2003; the plants will hurt during division propagation Ebrahim, 2004; Yip et al., 2006), shoots and created the wound can be easily infected (Cohen, 1981) and leaves (Gong Xue Qin et by several fungal and bacterial infections al., 2008; Zheng, 2010) have been used for during the cultivation practice. Growing of the dedifferentiation of the calla lily. At the calla lily has been studied broadly (Plummer et present time demand will need to be support al., 1990; Corr & Widmer, 1991), the major by high biomass yield varieties with improved efforts has been made to focused on the agronomical traits such as higher quality and control of flowering and tuber storage (Corr, quantity plant material production. Therefore Widmer, 1987, 1988). Commercially, this will generate the need for calla lily multiplication of calla lily through branches development through in vitro methods. The and tubers is applied to increase the size and purpose of this overview is to summarize the the number of tubers. However, there is existing literature for the in vitro culture of serious crop losses are caused by field-grown calla lily that may help to be acquainted from tubers infected by Erwinia soft rot (Chen et al., beginning to end tissue culture technology in 2000). Because of high infection in calla lily and provide a baseline for further individuals, Cohen (1981) and Ruiz (1996) improvement in the calla lily propagation with colleagues introduced plant propagation through in vitro techniques. through an in vitro method. The in vitro Conventional methods used for calla technique for long has been elaborated propagation and diseases commercially for the propagation of plant Calla lilies are conventionally multiplied material for agricultural, horticultural, through rhizomes commercially, annual pharmaceutical, or research purposes, and for multiplication of calla lily through offsets and building up disease-free plant materials tuber division is practiced to increase the (Thorpe, 2007). With the induction of number of tubers of flowering size. Several dedifferentiation is regulated by endogenous forms of rot can affect calla lilies which and exogenous growth regulators in the plant resulting in poor growth, wilting and possible nutrient medium (MS) (Murashige & Skoog, plant death. However, differentiation of field 1962). An important role in dedifferentiation grown tubers is subject to infection by erwinia stimulation is played by the type and con- soft rot, which causes serious losses in calla centration of plant growth regulators and their lily crop (Chen et al., 2000). Crown rot, root contact (Khanam et al., 2000). It has been rot, and pythium rot are caused by separate found that various combinations of auxins and different pathogens in poorly drained soil. cytokinins in the medium affected the strength Calla lilies are also affected by powdery of callus formation (Makunga et al., 2005). For mildew, armillaria rot, gray mold, blight, and

Kumar and Dogra Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 ISSN: 2582 – 2845 leaf spots. Spotted wilt and dasheen mosaic are In vitro tissue culture of calla lily two viruses that can attack calla lilies shows Plant regeneration from in vitro culture can be their effect on the leaf, flower stalks, and achieved by either organogenesis or petioles. embryogenesis. Tissue culture is used to introduce new cultivars and provide plant materials free from known viruses (Cohen, Plant tissue culture 1981; Yao et al., 1995). Tissue culture-derived Plant tissue culture is a technique used to plant material is proposed to be an alternative develop complete plantlets from a cell, tissue source of planting material for tuber or any other part of the plant when cultured on production in calla lily (Clemens & Welsh, a suitable medium under aseptic conditions. 1993; Cohen, 1981).

Organogenesis

Direct Indirect

Suspension Callus cell culture Shoot

Shoot Shoot

Root Root Root

Complete plantlet

Fig. 1: Micropropagation of plants by organogenesis

In vitro establishment of calla lily through cultured on MS media supplemented with tubers concentrations of BAP (0.5 to 5 mg dm-3), Danuta Kulpa., 2016 observed that the Culture KIN (0.5 to 5 mg dm-3), TDZ (0.1 to 1 mg dm- initiation of calla lily on MS media 3) and 2iP (2.5 to 15 mg dm-3) or BAP (0.5 to supplemented with 3 mg dm-3 BAP. During 7.5 mg dm-3) with IAA (0.5 to 2 mg dm-3). The multiplication stage, adventitious shoots was highest multiplication rate for Zantedeschia

Kumar and Dogra Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 ISSN: 2582 – 2845 was obtained on MS medium supplemented concentrations i.e; 5, 10, 25 and 50 μM. The with the concentration of BAP 2.5 mg dm-3. highest number of axillary shoots was Also MS medium with BAP of concentration observed on MS medium supplemented with 2.5 mg dm-3 shows the highest shoot length BA with a concentration of 25 and 50 μM. The (3.41 cm) and the number of adventitious treatment with BA 25 μM shows an increase in shoots (4.13). The highest rooting percentage the formation of rhizomes in the cultures. The was observed in MS medium fortified with 0.1 rooting was observed in all media used in the mg dm-3 IBA as compared to other rooting experiments. The highest rooting percentage hormones used. Effects of different sucrose was found in MS medium supplemented with concentrations in the culture medium on the BA of concentration 5 or 10 μM. growth of colored Zantedeschia in vitro under In vitro establishment of calla lily through

CO2 enrichment conditions the plantlets in shoot tips vitro of colored Zantedeschia had the largest The addition of auxins to the MS medium root number, root weight, and root vigor under supplemented with 8.87mM BA slightly 0g/l (sugar-free culture) treatment. And they enhanced multiple shoot formation in the had the largest plant height, leaf length, and explants. Multiplication of six cultivars of leaf chlorophyll content, but poor root vigor Zantedeschia genus comprising different under 3.0 g/l sugar (Wang et al., 2016). flower types and colors were tested and Ribeiro et al. (2009) observed that in vitro achieved using only one regeneration medium development of calla lily effect of sucrose and (MS + 8:87 mM BA + 2:46mM IBA). gibberellic acid concentrations. Plant tissue Different MS medium strength, air culture allows fast large-scale clonal temperature (15, 20, 25, and 30℃) and light propagation and provides healthy uniform quality fluorescent, red + blue, red and blue plant material. During the in vitro light provided by an LED (light-emitting micropropagation, the type of explant and diode) system were used (without concentrations of growth regulator could phytohormone) to stimulate in vitro shoot and influence the growth of seedlings. It was found root development. Half strength MS or MS that sucrose and GA3 concentrations in MS and cultures maintained at 25℃ were found to medium increase the efficiency of the in vitro be equally suitable for shoot tip culture of multiplication of calla lily. It was also found Zantedeschia albomaculata. Shoot elongation, that the addition of 60.5 g/ L sucrose and 5 as well as fresh and dry weight, was mg/L GA3 in MS medium shows the highest significantly increased when cultures were sprouts number. Whereas the higher length of kept under red or blue light (chang et al., the above-ground part the addition of 45.3 g/L 2002) Multiplication of six cultivars of sucrose and 10 mg/L GA3 was found most Zantedeschia genus comprising different effective. MS medium fortified with sucrose in species, flower types and colors were tested the range of 51.13 – 56.5 g/L showed the using only one regeneration medium (MS + highest root length and number of roots. 8:87mM BA + 2:46 mM IBA). All the Kozak et al., 2009 found that the effect of cultivars responded well and regeneration was benzyladenine on shoot regeneration in vitro observed among all the cultivars tested in the of Zantedeschia aethiopica ‘Green Goddess’. present experiments. However, the number of Shoots of Zantedeschia aethiopica ‘Green new shoots developed per explant and their Goddess’ obtained from aseptically grown fresh weight at 25d varied in different shoot clusters were used as the source of cultivars. Such differential responses among explant cultured in vitro on MS medium cultivars have already been reported in various supplemented with BAP of different plant species (Ahroni et al., 1997; Zuker et al.,

Kumar and Dogra Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 ISSN: 2582 – 2845 1997; Chakrabarty et al., 1999). However, the and 6.0 mg/L) combined with shoots that regenerated from shoot tip explants 0.1mg/L naphthalene-1-acetic acid (NAA), 3% on this medium had small leaves and a slow sucrose and 0.7% agar. CTT dye was used for growth rate. Bulblets and shoots of Narcissus quantifying cell viability of callus from nodal rooted only when the culture medium explants. Plantlets were individualized and contained MS salts at half strength (Seabrook inoculated with different concentrations of et al., 1976) and the response worsened with NAA and indol-3-butyric acid (IBA) (0.0, 1.0, increasing MS salt levels. The promoting 2.0 and 3.0 mg/L) for in vitro rooting. The effect of diluted mineral salt solution on highest shoot recovery occurred with 4.0 rooting is probably due to a reduced nitrogen mg/L BA combined with 0.1 mg/L NAA. The level (Sriskandarajah et al., 1990). Cultures highest percentage of rooted shoots (85%) was maintained at 25˚C showed the highest growth found in the culture medium with 2.0 rate as evidenced by the highest fresh weight, mg/L IBA. The acclimatization of rooted dry weight, leaf area, root number, and shoots in Plantmax® was successfully chlorophyll content. Chang et al., 2003 accomplished and 100% survival of plant reported that the micropropagation of calla lily material was observed during this stage. Our (Zantedeschia albomaculata) via in vitro shoot results demonstrate that micropropagation of tip proliferation onto Murashige and Skoog calla lily is successful using nodal explants (MS) medium supplemented with different (Nery et al. 2018). The medium used for plant growth regulator concentrations and explants was based on Murashige and Skoog combinations of the four cytokinins tested, 6- medium. Plantlets regenerated from explants benzyl adenine (BA) and thidiazuron (TDZ) were examined by electron microscopy. were found to be more effective. An effective Plantlets from basal parts of the petals and of concentration of BA (8.87 µM) or TDZ (4.54 basal pieces of leaves used as explants resulted µM) produced an average of 3.8 and 3.2 shoots in the best virus elimination. In conclusion, per explants but with the increasing tissue culture is applicable for virus concentrations of cytokinins often leads to elimination in lily cultivars and hybrids but the lower proliferation rate and slow growth. success depends on the type of explants. The Addition of auxins to the MS medium medium used for explants was based on supplemented with 8.87µM BA slightly Murashige and Skoog (1962; MS) medium: increasing multiplication of shoot formation in twice MS with addition of 1 mg/L 6-benzyl the explants. Multiplication of six cultivars of amino purine, 1 mg/L naphthaleneacetic acid Zantedeschia genus comprising different for basal pieces of the petals by Takayama and flower types and colors were tested and Misawa (1982); for bulbils from stem – with developed using only one regeneration addition of 5 mg/L naphthaleneacetic acid, 0.5 medium (MS + 8.87 µM BA + 2.46µM IBA). mg/L kinetin, 30 g/L sucrose and 10 g/L agar; Half strength MS or MS medium cultures MS with addition of 5 mg/L 6-benzyl amino maintained at 25˚C were found to be suitable purine, 0.1 mg/L naphthaleneacetic acid for for the shoot tip culture of Z. albomaculata. bulblets from scale by Jakobsone and In vitro establishment of calla lily through Andersone (1997) and MS with addition of 0.1 the leaf mg/L 6-benzyl amino purine, 1 mg/L Micropropagation and histological analysis of naphthaleneacetic acid for the basal part of Calla lily by transferring the leaf, nodal and leaves as explants by Niimi and Onozava root (1 cm2) explants to Murashige and Skoog (1979). All mother material and plantlets were medium (MS) supplemented with 6-benzyl examined by electron microscopy (Robinson adenine (BA) (0.0, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0 et al. 1987) and DAS ELISA (Hagita, 1989).

Kumar and Dogra Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 ISSN: 2582 – 2845 Vered et al., 2004 studied the hormonal in Zantedeschia plantlets in plant tissue culture control of inflorescence development in can act as an efficient model to study the role

plantlets of calla lily (Zantedeschia spp.) of GA3 and other factors in the flowering grown in vitro. Hormonal control of flower process of day-neutral plants that do not induction and inflorescence development in require external signals for flower induction vitro was studied in photo periodically day- under natural conditions. Jonytiene et al., 2017 neutral calla lily (Zantedeschia spp., colored studied that Factors affecting Zantedeschia

cultivars). The effect of gibberellins (GA3, Spreng dedifferentiation in vitro. callus 5.8–2900 µM) and the cytokinin (BA, 0.4– induction on somatic tissues of the arum lily, 13.3 µM) on inflorescence development were the most effective MS medium supplemented

studied in plantlets developed from plant tissue with 0.5 mg/L IAA + 2.0 mg/L BAP (leaf

culture. Plantlets were kept in GA3 and BA explants), 2.0 mg/L IAA + 2.0 mg/L BAP

solutions before replanting in new media. GA3 (spathe explants), and 1.0 mg/L IAA + 2.0

is essential for the shift from the vegetative to mg/L BAP (petiole explants) was used. the reproductive stage. Inflorescence Somatic tissues of the calla lily formed callus development from the apical bud was observed most intensively in the MS medium fortified

after 30–50 days in GA3 treated plantlets with 1.0 mg/L IBA + 2.0 mg/L BAP (leaf

grown in vitro, while BA did not show an explants), 1.0 mg/L IAA + 2.0 mg/L BAP

effect on flower induction. But in the presence (petiole explants), and 2.0 mg/L KIN (spathe

of GA3, BA at concentrations up to 4.4µM explants). Petiole segments explants formed shows enhanced inflorescence differentiation. callus more closely than isolated tissues of The results show that inflorescence developed leaves and spathe.

Table 1: In vitro micropropagation of calla lily

Type of explants Shooting media Rooting media References

Bulbils fromStem 5 mg/L BAP 0.1mg/L NAA Jakobsone and Andersone, 1997 Leaf 0.1 mg/L BAP 1 mg/L NAA Niimi and Onozava, 1979 Leaf 1 mg/L BAP 1 mg/L NAA Takayama and Misawa, 1982 Leaf 0.1mg/l BAP 1mg/l NAA Niimi and Onozava , 1979 Leaf 0.5 mg/L IAA + 2.0 mg/L BAP _ Jonytiene et al., 2017 Nodal 4.0 mg L-1 BA + 0.1 mg L-1 NAA 2.0 mg L-1 IBA Nery et al., 2018 Petiole 1.0 mg/L IAA + 2.0 mg/L BAP _ Jonytiene et al., 2017 Shoots 25 Μm BA BA 5 or 10 Μm BA Kozak et al., 2009 Shoots 8:87 mM BA + 2:46mM IBA _ chang et al., 2003 Spathe 2.0 mg/L KIN _ Jonytiene et al., 2017 Tubers 2.5 mg dm-3 BAP 0.1 mg dm-3 IBA Danuta Kulpa, 2016

Tubers 10 mg/L GA3 51.13 – 56.5 g/L sucrose Ribeiro et al., 2009

Kumar and Dogra Ind. J. Pure App. Biosci. (2020) 8(2), 144-153 ISSN: 2582 – 2845

A B

Fig. 2: Calla lily tuber on MS medium supplemented with different growth regulators (A) Shoot establishment (B)

CONCLUSION Chakrabarty, D., Mandal, A. K. A., & Datta, S. Calla lily is a commercially important crop for K. (1999). Management of chimera cut flower production as well as medicinal through direct shoot regeneration from property gaining worldwide. Lack of quality florets of chrysanthemum planting material is a major drawback in large- (Chrysanthemum morifolium Ramat.), scale cultivation of calla lily. Tissue culture Journal of Horticulture Science techniques proved to be boon for the Biotechnology 74, 293–296. production of high quantity and quality Chang, H. S., Charkabarty, D., Hahn, E. J., & planting material for farmers. At present, Paek, K. Y. (2003). Micropropagation direct regeneration of plantlets via adventitious of calla lily (Zantedeschia al- shoot and tuber explants is considered as a bomaculata) via in vitro shoot tip preferred method for calla lily plant proliferation, In vitro Cell regeneration. Future research emphasized on Development Biology 39(2), 129–34. the development of protocols for direct Chang, S. H., Chakrabarty, D., Hahn, J. E., & regeneration of shoot buds from leaf explants, Paek, Y. K. (2003). Micropropagation protocols for regeneration through somatic of calla lily (zantedeschia embryo-genesis need to be improved to albomaculata) via in vitro shoot tip develop as it can help in producing true to type proliferation, In vitro Cell and and homozygous plants with improved quality Development Biology 39, 129–134. and development of improved genotypes with Chen, J. J., Liu, M. C., & Ho, Y. H. (2000). a high medicinal value content, higher biomass Size of in vitro plantlets affects production, wider adaptability, viable seed subsequent tuber production of ac- production. climated calla lily, Horticulture Science 35, 290–298. REFERENCES Clemens, J. & Welsh, T. E. (1993). An Ahroni, A., Zukur, A., Rozen, Y., Shejtman, overview of the New Zealand calla H., & Vain Stein, A. (1997). An industry, research direction and year- efficient method for adventitious shoot round tuber production, Acta regeneration from stem segment Horticulture 337, 161-166. explants of gypsophila, Plant Cell Clemens, J. D., Dennis, D. J., Butler, R.C., Tissue and Organ Culture 49, 101– Thomas, M. B., Ingle, A., & Welsh, T. 106. E. (1998). Mineral nutrition of Copyright © March-April, 2020; IJPAB 150

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