The application of leafy explant micropropagation protocol in enhancing the multiplication ef ciency of Alstroemeria

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1 Scientia Horticulturae 85 (2000) 307±318 The application of leafy explant micropropagation protocol in enhancing the multiplication ef ciency of Alstroemeria Hsueh-Shih Lin 1, Marjo J. De Jeu *, Evert Jacobsen Graduate School of Experimental Plant Sciences, Laboratory of Plant Breeding, Wageningen University, PO Box 386, NL-6700 AJ Wageningen, Netherlands Accepted 22 December 1999 Abstract Six tetraploid Alstroemeria clones were micropropagated by rhizome multiplication, a system whereby the shoots were cut off from the rhizome and discarded. In a three-week subculture interval, the average rhizome multiplication rate for all genotypes was 2.3. In one selected genotype the discarded shoots were used to initiate another micropropagation system: the leafy explant micropropagation system. In this system the leaves with axils and stem tissue were cut off the discarded shoots and cultured for 10 days in shoot induction medium followed by subculture on shooting medium for the formation of adventitious shoots. From the discarded shoots of one rhizome explant, on an average 1.1 leafy explants could be excised. Thus, combining the rhizome micropropagation together with the leafy explant micropropagation, the total number of explants were enhanced twice. Regenerated plants from both micropropagation systems were grown in the greenhouse to maturity. Plants regenerated from leafy explants were morphologically identical to those originated from the rhizome micropropagation. The leafy explant micropropagation protocol was not only suitable for in vitro grown explants of different genotypes, but also applicable to in vivo grown explants. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Alstroemeria; Inca lily; Micropropagation; Monocots; Rhizome * Corresponding author. Tel.: ; fax: address: marjo.dejeu@users.pv.wau.nl (M.J. De Jeu) 1 Present address: Hualien District Agricultural Improvement Station, No. 150, Chi-an Road, sec. 2, Hualien, Taiwan, ROC /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 308 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± Introduction Alstroemeria is a perennial crop mainly cultured in greenhouses for cut ower production. It is a monocotyledonous cut ower that is vegetatively propagated by splitting of the rhizomes. This results in a rather low multiplication rate whereas seasonal restrictions occur. To solve these limitations several in vitro micropropagation systems have been developed, based on rhizome tip and/or rhizome meristem micropropagation (Gabryszewska and Hempel, 1985; Lin and Monette, 1987; Hakkaart and Versluijs, 1988; Pierik et al., 1988; Van Zaayen et al., 1992; Bond and Alderson, 1993; Gabryszewska, 1995). In a four-week subculture interval the rhizome multiplication rate is between 2.0 and 3.0, depending on culture conditions and the genotype (Pierik et al., 1988). Compared to other plant species this rate is rather low. Since the rhizome tip is found to be the best explant in Alstroemeria (Lin and Monette, 1987) and other organs are dif cult to regenerate into plants, the shoots usually are cut off and discarded during subculture. Lin et al. (1997) have developed a new micropropagation protocol by using leafy explants. In this protocol adventitious shoots are initiated at the stem epidermis on the leaf axil side, which develop into complete plants (Lin et al., 1997, 1998). This protocol provides an additional choice for micropropagation. It is expected that a combination of rhizome micropropagation and leafy explant micropropagation at the same time will enhance the multiplication ef ciency. Another aspect is the initiation of the rhizome micropropagation from an in vivo grown greenhouse plant. For this purpose usually the underground grown rhizome tip is used, whereby contamination is a major problem that is dif cult to overcome (Pierik et al., 1988; Pedersen and Brandt, 1992). The use of leaves as explants may solve this problem, because it is expected that disinfecting aerial plant tissue can be easier than disinfecting an underground grown plant part. In the present report a cyclic micropropagation system based on leafy explants is presented. The combination of two micropropagation systems, one based on rhizome explants and one based on leafy explants, is carried out. Morphological traits of owering plants originated from both micropropagation systems are compared. Micropropagation through leafy explants taken from in vitro and in vivo grown plants of an existing cultivar is also presented. 2. Materials and methods 2.1. Micropropagation based on rhizome explants Selfed seeds of VV024 (a tetraploid breeding line from Van Staaveren BV, Netherlands) were surface sterilized (rinsed 20 min in 1.5% sodium-hypochlorite

3 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± solution and then three times in sterilized water) and incubated on semi-solid half strength MS (Murashige and Skoog, 1962) medium with 1% sucrose for germination (Lin et al., 1997). After germination, the seedlings were grown on full strength MS medium with 3% sucrose and 0.3% gelrite for four months in subcultures. Thereafter the plantlets were transferred to MS medium supplemented with 0.5 mg/l BAP (BA 0.5), 3% sucrose and 0.3% gelrite, and constantly subcultured in a three-week interval for rhizome formation. Two months after subculture on BA 0.5 medium, many rhizomes were formed on each plantlet and these were used for the multiplication experiments. In order to trace the multiplication behavior of this selfed population, each individual plantlet was subdivided into many parts. Each part had a main rhizome and some aerial shoots and axillary buds on the rhizome. After cutting off the aerial shoots, the axillary buds on the rhizomes were kept and they were transferred into test tubes with BA 0.5 medium individually. Six progenies were selected and three plantlets (replicates) from each progeny were used in this experiment. Number of shoots and lateral rhizomes, originating from one rhizome explant were counted both before and after three-week subculture in order to calculate the multiplication rate. All the in vitro cultures were placed at 188C and 12 h light (Philips 32 W, 84 HF, 4000 lx). From one selected genotype (VV2406) 20 rhizomes were transferred to MS medium with 3% sucrose, 0.3% gelrite, 0.5 mg/l NAA and 0.4 g/l caseine hydrolysate in order to induce root formation. After one-month culture on this root-inducing medium, the plants were transferred to the greenhouse for further culture to owering Micropropagation based on leafy explants and development of a cyclic micropropagation system One Alstroemeria clone VV2406 was selected out of the selfed progenies of VV024 and micropropagated by rhizome explants for several months. Shoots of approximately 5 cm in length with three fully developed leaves were collected and shoot apices were removed. From each shoot with three young leaves three leafy explants were cut according to the leafy explant micropropagation protocol described by Lin et al. (1997). Ten leafy explants were cultured in one petri dish and at least 10 replicates were prepared for each experiment. After 10 days on induction medium and eight weeks on shooting medium (both under dark conditions) the leafy explants with developing adventitious shoots were transferred to test tubes containing BA 0.5 medium and were placed under 12 h light conditions (Philips 32 W, 84 HF, 4000 lx). The subculture interval was four weeks. For the development of a cyclic micropropagation system, newly formed shoots of about 5 cm in length were collected and the youngest three leafy explants were excised again, and cultured

4 310 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318 on induction medium for the next culture cycle. In total three cyclic cultures were performed in our experiment. Twenty plants regenerated from this cyclic micropropagation system were transferred to the greenhouse after the formation of well-developed rhizomes, shoots and roots. Morphological traits were measured and analyzed during the owering period. The performances of plants derived from rhizome micropropagation and leafy explant micropropagation were compared Combination of rhizome- and leafy explant micropropagation In the selected genotype VV2406 both rhizome and leafy explant micropropagation were applied during four subcultures of three-weeks each. Leafy explants were excised from the discarded shoots of the rhizome micropropagation system in order to measure the potential number of leafy explants available when both micropropagation methods were combined together Leafy explants cut from in vitro and in vivo grown plants Alstroemeria CV 118 plants (a tetraploid cultivar from Van Staaveren BV, The Netherlands) were subcultured in vitro on BA 0.5 medium, and were used for collecting shoots. The erect shoots of growth-chamber grown (in vivo) CV 118 plants were also collected. In order to compare the adventitious shoot formation ability of leafy explants from both in vitro and in vivo grown plants, leafy explants were excised and cultured on media according to the same procedures as mentioned above. The in vivo grown shoots were sterilized by 3% of sodium-hypochlorite for 15 min and rinsed three times in sterilized water before excision of the leafy explants. The three fully developed top leaves were used in all preparations. Depending on the size, either 10 leaves (in vitro) or 5 leaves (in vivo) were incubated in one petri dish and 5 replicates were prepared. Once the rhizome, shoots and roots were formed, the plants were transferred to the greenhouse for culture to owering. 3. Results 3.1. Rhizome micropropagation The rhizome explant was a stem-like, horizontally growing structure with 2±3 nodes and a sharp apex. Each node consisted of an upright growing shoot bud with an enlarged base. The rhizome apex, which appeared at one side of the former enlarged shoot base, was wrapped by a scale leaf and looked like a shoot

5 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± Fig. 1. Rhizome plant of Alstroemeria VV2406 micropropagated in vitro. FSL: rst scale leaf; NRA: newly formed rhizome apex; R: root; RA: rhizome apex; S: shoot; Barˆ0.5 cm. bud. One week after subculture on BA 0.5 medium, the rhizome apex was elongated and formed an upright growing bud. Two weeks later, this bud developed into a shoot with an enlarged base. Simultaneously, a new rhizome apex appeared at one side of the shoot base. On the opposite site of the rhizome apex a root developed in the medium (Fig. 1). The newly formed rhizome apex looked like an axillary bud of the rst scale leaf of the upright growing shoot. At the other nodes, one bud developed into a shoot and, in some cases, an axillary bud was formed at the other side of the shoot base. The axillary bud elongated and became a lateral rhizome tip subsequently. After three-week subculture the test tubes were lled with shoots and rhizome tips. The results of shoot and rhizome micropropagation in the six progenies of clone VV024 are shown in Table 1. On an average, each explant produced 4.2 new shoots and 2.1 lateral rhizomes. A large difference of the mean number of shoots per explant was found among the progenies, ranging from 2.7 to 6.4. However, the mean number of lateral rhizomes per explant among the different progenies ranged from 1.3 to 2.5. On an average, an original rhizome explant produced twice as much shoots than the lateral rhizomes in three-week intervals (Table 1).

6 312 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318 Table 1 In vitro multiplication rates of six selfed progenies of VV024 after rhizome micropropagation a Progeny No. of shoots per explant No. of lateral rhizomes per explant Multiplication rate Shoot Rhizome VV VV VV VV VV VV Average a Data were collected after three-week subculture of the rhizome explants on MS (1962) medium supplemented with 0.5 mg/l BAP. The average multiplication rate of shoots and rhizomes in the population was 3.0 and 2.3, respectively, based on a three-week interval, but a large variation was found between the progenies (Table 1). The multiplication rate of shoots did not associate with that of the rhizomes, e.g. the progeny VV2454 had the largest shoot multiplication rate but the smallest rhizome multiplication rate Cyclic micropropagation system using leafy explants In the leafy explant micropropagation system adventitious buds were initiated at the region between leaf base and stem node (leaf axil) after 10 days on induction medium followed by four weeks on shooting medium under dark conditions. After another four weeks on shooting medium, the buds developed into shoots and these were transferred to test tubes. The shoots kept growing in the test tubes and elongated. Two months later a rhizome apex was initiated at the rst node of one shoot. Once the rhizome was formed, individual shoots could be separated from the original leafy explant. Generally, when a leafy explant has ve or more adventitious shoots, separation into two surviving plantlets is possible. With an average of 5.3 shoots per explant, one leafy explant generates only one plantlet with rhizome and shoots. After subculture for another month, the rhizome developed into a complete plant with new shoots and roots. From a leafy explant to a newly formed rhizome, a regeneration cycle was completed. One cycle took about 4±5 months. Once the newly formed rhizomes started producing new axillary shoots, the old shoots gradually turned into yellow and died. Before yellowing, the old shoots were cut off and leafy explants could be used for the next micropropagation cycle. The second cycle was initiated by culturing the leafy explants excised from the rst cycle's shoots.

7 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± Table 2 Three cycles of adventitious shoot regeneration in Alstroemeria clone VV2406 by application of leafy explant micropropagation protocol Cycle Percentage of regeneration (%) Number of shoots/explant Average The results of the leafy explant micropropagation system during three cycles are presented in Table 2. The mean percentage of shoot regeneration ranged from 84 to 90% and there were no signi cant differences among the three culture cycles. The mean number of shoots per explant ranged from 4.9 to 5.9 and no signi cant differences were found. During the three micropropagation cycles the leafy explants always maintained high regeneration ability Combination of rhizome- and leafy explant micropropagation in one system In genotype VV2406 four subcultures of rhizome micropropagation were done and the suitable leaves were excised from the discarded shoots. From the in total 437 rhizome explants 500 suitable leafy explants could be excised. So, on an average 1.1 leafy explant per rhizome explant could be used during the rhizome micropropagation system Comparison of morphological traits of plants originated from rhizome- and leafy explant micropropagation In total 40 plants of clone VV2406, which were micropropagated either by rhizomes (20 plants) or by leafy explants (20 plants), were transferred to the greenhouse, when they had formed roots (after one month on root-inducing medium). Nineteen plants from rhizome micropropagation survived and 15 from the leafy explant micropropagation. The plants originating from rhizome micropropagation were larger in size than those from leafy explant micropropagation having a higher percentage of survival (95±75%) and more shoots per plant (Table 3, Fig. 2). The rhizome micropropagated plants started to ower eight months after transfer to the greenhouse. However, two weeks later the leafy explant micropropagated plants started to ower. Several morphological traits were measured and listed in Table 3. All the vegetative and generative traits we scored showed that plants micropropagated by the two different methods were morphologically identical (Fig. 2).

8 314 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318 Table 3 Comparison of morphological traits of Alstroemeria VV2406 plants, which were micropropagated by two different systems: rhizome micropropagation and leafy explant micropropagation a Traits Rhizome micropropagation Leafy explant micropropagation Vegetative parts No. of shoots/plant Plant height (cm) Leaf length (cm) Leaf width (cm) No. of leaves/shoot Generative parts No. of flowers/shoot No. of peduncles/shoot Peduncle length (cm) Flower length (cm) Flower width (cm) Flower color Pink Pink No. of anthers/flower 6 6 Anther color Yellow Yellow Pollen grain color Yellow Yellow a The data were collected eight months after transfer to the greenhouse. In total 19 plants from rhizome micropropagation and 15 plants from leafy explant micropropagation were measured. Fig. 2. Flowering of Alstroemeria plant VV2406, eight months after transfer to the greenhouse: (A) originated from rhizome micropropagation, (B) originated from leafy explant micropropagation.

9 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± Table 4 Adventitious shoot regeneration from different leafy explant sources of the tetraploid Alstroemeria CV 118 Explant source Percentage of regeneration Number of shoots/explant In vitro a In vivo b a Leafy explants were taken from in vitro grown plants, which were originated from rhizome micropropagation. b Leafy explants were taken from growth-chamber grown in vivo plants. Fig. 3. Leafy explant micropropagated plants of Alstroemeria CV 118 are owering in the greenhouse. The initial leafy explants were taken from either in vitro (left), or in vivo grown plants (right).

10 316 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± The application of leafy explant micropropagation in vitro or in vivo grown shoots The procedure of adventitious shoot formation on leafy explants of CV 118 was similar to that of VV2406. Although the size of isolated leafy explants cut from in vivo grown plants were larger than that of in vitro grown plants, there were no signi cant differences between those two types of explants in the percentage of shoot regeneration and the number of shoots per explant (Table 4). Compared to the leafy explant micropropagation in VV2406 (Table 2) the percentage of regeneration of the rst three leaves of CV 118 was about half of that of VV2406, 43% compared to 88%. Leafy explants together with regenerating shoots were subcultured in test tubes containing BA 0.5 medium and after two months the rhizomes were formed at the shoot base. It took ve months from starting the experiment to obtain complete plants with shoots and rhizomes. Once the rhizomes were formed, the plants were separated from the original leafy explants and subcultured on root-inducing medium. The complete plants including rhizome, shoots and roots were transferred to the greenhouse and they started owering about six months later. Visual comparison was made and there were no morphological differences found between the plants derived from either in vitro or in vivo grown plants (Fig. 3). 4. Discussion Results of our rhizome micropropagation experiments are comparable to previous reports on multiplication of rhizome tips of Alstroemeria hybrids (Pierik et al., 1988). The differences between the shoot and rhizome multiplication rate over progenies suggest a genotypic effect, due to the expected genetic variation of the selfed progeny. The rhizome is the main propagation organ in Alstroemeria. The aerial shoots grow out of the rhizome node and axillary rhizome buds are present at the base of each aerial shoot. Bond and Alderson (1993) suggested that a high apical dominance of the rhizome apex or of aerial shoots is responsible for the suppression of the formation of lateral rhizomes. They found that the number of shoots often was not associated with the number of rhizomes. We also found this in our results, because in all tested progenies, the number of shoots per plant was not equal to the number of lateral rhizomes. In our experiment we used two different genotypes for the leafy explant micropropagation: VV2406 (breeding line) and CV 118 (cultivar). The response of both genotypes was different when we compare the leafy explant micropropagation applied in the in vitro grown leaves: 88% of regeneration of the rst three leaves in VV2406 and 43% of regeneration of the rst three leaves

11 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307± in CV 118. Although the response in the cultivar was lower than that in the breeding line, it seems that the leafy explant micropropagation can be applied in different genotypes of Alstroemeria. We used the same method and chemicals in both genotypes. May be each genotype acquires an adapted treatment in order to increase the percentage of regeneration. The time needed from transfer to the greenhouse to owering was longer for the leafy explant micropropagated plants than for the rhizome micropropagated plants. This delay is probably not due to the different propagation methods, but to the different sizes of the in vitro plant. Pedersen et al. (1996) found that plant size might in uence the time to owering in Alstroemeria and the larger the size, the earlier the owering. In our experiments, the ex vitro plants produced by rhizome micropropagation were larger than those regenerated from leafy explants, which might be an explanation for earlier owering. The rhizome tip was previously found to be the only explant for initiating the in vitro micropropagation system in Alstroemeria (Lin and Monette, 1987; Gabryszewska, 1995). Since the plants were grown in the soil, sterilizing of underground rhizomes became a major problem and the contamination due to internally present micro-organisms was very dif cult to overcome (Pierik et al., 1988). Thus, Pedersen and Brandt (1992) recommended a method for disinfecting rhizome tips that contained two disinfectants and three disinfecting steps. Lin et al. (1997) reported a micropropagation system by using in vitro grown leafy explants (including stem node) as initial plant material. In this paper we reported that this leafy explant micropropagation system was also useful by using in vivo (growth-chamber) grown leafy explants. Leafy explants, taken from aerial shoots, were easier to sterilize than underground rhizomes. Besides, some other advantages of this method were found: no damage of the rhizomes, less disinfecting steps, less contamination problems and the explants were easier to collect. The conventional way of micropropagation for Alstroemeria is based on rhizomes. Therefore, the shoots are of no use and will be cut off and discarded during each subculture. This report has shown that both rhizomes and leafy explants can be used for micropropagation of Alstroemeria. When combining both micropropagation systems together, each rhizome explant delivers 1.1 suitable leafy explant for culture. With an average regeneration percentage of 88% in the leafy explant micropropagation, each rhizome explant produces one extra plant through leafy explant micropropagation. This means that the number of explants is doubled in the three-week interval. Although the period in which a leafy explant delivers complete plants is longer (4±5 months instead of two months) the multiplication ef ciency is enhanced. However, the combination of the two techniques did not deliver the number of explants we should have expected according to the experiments of the separated micropropagation techniques. In our research a rhizome explant of Alstroemeria clone VV2406

12 318 H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318 produced 2.2 rhizomes and 4.2 shoots for every three-week interval (Table 1). Each shoot could theoretically produce three leafy explants with an average regeneration capacity of 88% (Table 2). We only could use 1.1 leafy explant per rhizome explant in our experiment may be due to the dif culty of cutting the individual leaves because of the very short stem internodes produced during the rhizome micropropagation method. In the combination of the two micropropagation methods we could adjust the rhizome micropropagation method by concentrating on the development of well-formed shoots carrying three leaves suitable for leafy explant micropropagation. By doing so we expect a large enhancement of the multiplication ef ciency in Alstroemeria. Acknowledgements The Alstroemeria stock plants (VV024 and CV 118) were kindly supplied by the breeding company Van Staaveren BV, The Netherlands. Hong-Gi Jang took part in the research during his sabbatical leave at Wageningen University. This research was kindly nanced by the National Science Council, Taiwan, ROC. References Bond, S., Alderson, P.G., The in uence of apical dominance on the in vitro multiplication of the rhizome of Alstroemeria. J. Hort. Sci. 68, 905±910. Gabryszewska, E., Plant regeneration of Alstroemeria in vitro. Acta Agrobotanica 48, 95± 104. Gabryszewska, E., Hempel, M., The in uence of cytokinins and auxins on Alstroemeria in tissue culture. Acta Hort. 167, 295±300. Hakkaart, F.A., Versluijs, J.M.A., Virus elimination by meristem tip culture from a range of Alstroemeria cultivars. Neth. J. Plant Pathol. 94, 49±56. Lin, H.S., De Jeu, M.J., Jacobsen, E., Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L. Plant Cell Rep. 16, 770±774. Lin, H.S., De Jeu, M.J., Jacobsen, E., Formation of shoots from leaf axils of Alstroemeria: the effect of the position on the stem. Plant Cell Tissue Organ Cult. 52, 165±169. Lin, W.C., Monette, P.L., In vitro propagation of Alstroemeria `Alsaan'. Plant Cell Tissue Organ Cult. 9, 29±35. Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473±497. Pedersen, C., Brandt, K., A method for disinfection of underground rhizome tips of Alstroemeria and Heliconia. Acta Hort. 325, 499±504. Pedersen, C., Hansen, C.W., Brandt, K., Kristiansen, K., Alstroemeria plantlets can be induced to owering by cold treatment during in vitro culture. Sci. Hort. 66, 217±228. Pierik, R.L.M., Van Voorst, A., Booy, G., Van Acker, C.A.M., Lelivelt, C.L.C., De Wit, J.C., Vegetative propagation of Alstroemeria hybrids in vitro. Acta Hort. 226, 81±89. Van Zaayen, A., Van Eijk, C., Versluijs, J.M.A., Production of high quality, healthy ornamental crops through meristem culture. Acta Bot. Neerland. 41, 425±433.