Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol85.Issue4.Agust2000:
Scientia Horticulturae 85 (2000) 307±318
The application of leafy explant micropropagation
protocol in enhancing the multiplication
ef®ciency of Alstroemeria
Hsueh-Shih Lin1, 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.: 31-317-482838; fax: 31-317-483457.
E-mail address: [email protected] (M.J. De Jeu)
1
Present address: Hualien District Agricultural Improvement Station, No. 150, Chi-an Road, sec.
2, Hualien, Taiwan, ROC.
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 2 5 - 4
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H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
1. 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
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
309
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.
2.2. 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
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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.
2.3. 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.
2.4. 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
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
311
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; Bar0.5 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).
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Table 1
In vitro multiplication rates of six selfed progenies of VV024 after rhizome micropropagationa
Progeny
No. of shoots
per explant
No. of lateral rhizomes
per explant
Multiplication rate
Shoot
Rhizome
VV2406
VV2410
VV2434
VV2435
VV2452
VV2454
4.20.8
2.71.1
4.30.5
6.41.7
4.02.0
3.82.1
2.20.3
1.30.6
2.51.3
2.20.4
2.22.2
2.01.0
2.50.5
2.71.1
3.60.8
2.30.7
3.22.2
3.82.0
1.90.3
2.01.0
3.31.7
2.10.9
2.81.5
1.50.5
Average
4.2
2.1
3.0
2.3
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.
3.2. 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.
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
313
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
1
2
3
8412.1
8910.5
9111.6
5.91.4
4.90.8
5.01.2
Average
88
5.3
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.
3.3. 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.
3.4. 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).
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Table 3
Comparison of morphological traits of Alstroemeria VV2406 plants, which were micropropagated
by two different systems: rhizome micropropagation and leafy explant micropropagationa
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
14.22.1
40.65.2
6.40.6
1.00.1
17.01.8
11.73.0
43.53.4
6.20.7
1.10.1
19.33.7
Generative parts
No. of flowers/shoot
No. of peduncles/shoot
Peduncle length (cm)
Flower length (cm)
Flower width (cm)
Flower color
No. of anthers/flower
Anther color
Pollen grain color
12.82.7
5.10.5
7.91.5
5.10.3
4.60.4
Pink
6
Yellow
Yellow
11.42.3
4.60.5
7.00.8
5.10.3
4.40.4
Pink
6
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.
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
315
Table 4
Adventitious shoot regeneration from different leafy explant sources of the tetraploid Alstroemeria
CV 118
Explant source
a
In vitro
In vivob
Percentage of regeneration
Number of shoots/explant
42.810.0
37.83.9
3.41.0
5.23.2
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).
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H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
3.5. 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
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
317
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
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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.
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The application of leafy explant micropropagation
protocol in enhancing the multiplication
ef®ciency of Alstroemeria
Hsueh-Shih Lin1, 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.: 31-317-482838; fax: 31-317-483457.
E-mail address: [email protected] (M.J. De Jeu)
1
Present address: Hualien District Agricultural Improvement Station, No. 150, Chi-an Road, sec.
2, Hualien, Taiwan, ROC.
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 2 5 - 4
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H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
1. 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
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
309
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.
2.2. 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
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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.
2.3. 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.
2.4. 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
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
311
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; Bar0.5 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).
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Table 1
In vitro multiplication rates of six selfed progenies of VV024 after rhizome micropropagationa
Progeny
No. of shoots
per explant
No. of lateral rhizomes
per explant
Multiplication rate
Shoot
Rhizome
VV2406
VV2410
VV2434
VV2435
VV2452
VV2454
4.20.8
2.71.1
4.30.5
6.41.7
4.02.0
3.82.1
2.20.3
1.30.6
2.51.3
2.20.4
2.22.2
2.01.0
2.50.5
2.71.1
3.60.8
2.30.7
3.22.2
3.82.0
1.90.3
2.01.0
3.31.7
2.10.9
2.81.5
1.50.5
Average
4.2
2.1
3.0
2.3
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.
3.2. 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.
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
313
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
1
2
3
8412.1
8910.5
9111.6
5.91.4
4.90.8
5.01.2
Average
88
5.3
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.
3.3. 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.
3.4. 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).
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Table 3
Comparison of morphological traits of Alstroemeria VV2406 plants, which were micropropagated
by two different systems: rhizome micropropagation and leafy explant micropropagationa
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
14.22.1
40.65.2
6.40.6
1.00.1
17.01.8
11.73.0
43.53.4
6.20.7
1.10.1
19.33.7
Generative parts
No. of flowers/shoot
No. of peduncles/shoot
Peduncle length (cm)
Flower length (cm)
Flower width (cm)
Flower color
No. of anthers/flower
Anther color
Pollen grain color
12.82.7
5.10.5
7.91.5
5.10.3
4.60.4
Pink
6
Yellow
Yellow
11.42.3
4.60.5
7.00.8
5.10.3
4.40.4
Pink
6
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.
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
315
Table 4
Adventitious shoot regeneration from different leafy explant sources of the tetraploid Alstroemeria
CV 118
Explant source
a
In vitro
In vivob
Percentage of regeneration
Number of shoots/explant
42.810.0
37.83.9
3.41.0
5.23.2
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).
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H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
3.5. 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
H.-S. Lin et al. / Scientia Horticulturae 85 (2000) 307±318
317
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
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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.
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