Somaclonal Variation in in vitro Regenerated Ledebouria graminifolia (Hyacinthaceae), an Indigenous Bulb in Botswana and its Potential Exploitation as an Ornamental Plant
The aim of this study is to report on the occurrence
of somaclonal variation in in vitro regenerated Ledebouria graminifolia
an indigenous bulb in Botswana and assess its potential use as an ornamental
plant. Plants that were regenerated using tissue culture were planted
in a mixture of garden soil and potting soil in the greenhouse and also
grown in the field under natural conditions without additional resources
supplied to the plants. Morphological features and tolerance to drought
were used to assess somaclonal variation among clones. In vitro
propagated plants grew well in the greenhouse, they produced large attractive
leaves and many flowered continuously. Field grown plants acclimatized
well and survived periods of droughts and high temperatures. Morphological
evaluation of the plants showed five visually distinct variants. Morphotype
1 consisted of prostrate plants with large, fewer, bright green leaves.
Morphotype 2 consisted of prostrate plants with large, less bright green
lanceolate leaves. Morphotype 3 consisted of plants with medium aristate,
grayish green, curly leaves with rubbery appearance. Morphotype 4 had
intermediate characteristics between 2 and 3 with grayish, lanceolate
leaves. Morphotype 5 typically represented the parental phenotype of wild
plants, consisted of very erect plants with curly, linear leaves with
rubbery appearance. The variants also showed differences in their tolerance
to drought with morphotypes 3 and 5 being most tolerant and morphotypes
1and 2 the least tolerant. The bulb could be mass propagated and exploited
for ornamental purposes in Botswana to complement or as alternatives to
popular exotic plants currently dominating the floriculture industry.
to cite this article:
D.D. Shushu, J.M. Comar and B.M. Abegaz, 2009. Somaclonal Variation in in vitro Regenerated Ledebouria graminifolia (Hyacinthaceae), an Indigenous Bulb in Botswana and its Potential Exploitation as an Ornamental Plant. Journal of Biological Sciences, 9: 152-158.
In vitro propagation has great potential for propagating vegetatively
reproducing plants and has been proposed to be a biotechnological tool which
offers potential solution to problems of indigenous medicinal and ornamental
plants over-exploitation in many parts of Africa (Afolayan
and Adebola, 2004). Mass propagation using tissue culture techniques could
offer cheaper alternatives to harvesting from the wild as is the common practice
in many African countries and also contribute to the conservation of indigenous
useful plants (McCartan and Van Staden, 1999; Entwistle
et al., 2002; Afolayan and Adebola, 2004).
Plants that have been regenerated using tissue culture methods often show variation
(Sahijram et al., 2003; Podwyszynska,
2005). This variation, called somaclonal variation (Larkin
and Scowcroft, 1981; Scowcroft and Larkin, 1988) has
been found to have a genetic base and can be transmitted to the progeny through
sexual reproduction or vegetative propagation. Plant tissue culture, can therefore
provide additional genetic variability rapidly using simple technology and can
provide an additional tool for plant improvement (Evans and Sharp, 1986; Bajaj,
1990; Karp, 1995; Jain and De
Klerk, 1998; Tremblay et al., 1999) and a
comprehensive review on propagation of ornamental plants using tissue culture
is given by Rout et al. (2006).
Botswana is mostly a semi arid and arid country rich in diversity of indigenous
plants adapted to drought conditions. The plants and their products are important
in sustaining life in rural communities and many have high export potential
(Taylor, 1985). Plants that are harvested from the wild
are mainly leafy vegetables, fruits and medicinal plants (Mateke
and Matlhare, 2000; Rositter et al., 1997)
and sold in markets both locally and outside the country and have played
a big part in supplementing income to most rural communities. In Botswana, collection
and marketing of these indigenous plants has become very popular comparable
to street-vended foods, especially in large towns. While there is a growing
number of plant nursery business and street plant vendors in the country, there
is little effort to promote the exploitation/use of indigenous plants as ornamental
plants despite the growing awareness and interest in a green urban environment.
Most of the plant species currently being exploited as ornamental plants for
house and gardens are exotics and few indigenous succulents. Where, they are
used, the indigenous plants are often undervalued in favour of exotics and aliens.
However, there are many non-succulent plants with great potential that could
be used in the floriculture industry. In addition, the plants that are currently
collected from the wild have great potential for multipurpose uses. The underutilized
plants could be exploited in the floriculture industry and can contribute significantly
to the livelihoods of communities by providing additional income especially
to rural and urban women, who are the driving force behind the booming informal
economic sector in Botswana.
There are many advantages of using indigenous plants as ornamentals compared
to exotic or alien species. Indigenous plants are usually well adapted to local
arid conditions and increasing shortage of rains and irrigation water than most
exotics. The rural communities, particularly women, are familiar with the plants
and usually have potential multiple uses. Hence indigenous plants could fill
in an important gap within the horticultural industry (Mander
et al., 1996). Therefore there is a need to create awareness of these
advantages and promote the exploitation of indigenous plants for ornamental
purposes so as to offer an alternative source of income generation especially
to rural women and to empower them with new ideas of the potential multipurpose
uses and not just harvest plants for medicinal use; but also in propagation,
multiplication and for biodiversity conservation.
The aim of this study is to evaluate the performance of in vitro regenerated
Ledebouria graminifolia plants in the green house and their acclimatization
to field conditions, so as to promote its sustainable exploitation as an ornamental
plant and for the conservation of the plant. Ledebouria graminifolia,
is an indigenous bulb in Botswana with great potential as an ornamental plant
for indoors and outdoors. The bulb is extensively harvested from the wild and
sold on markets for its medicinal properties (Mutanyatta
et al., 2003; Shushu et al., 2005).
MATERIALS AND METHODS
This study was conducted at the University of Botswana, Department of Biological
Sciences in 2004-2007. The plants used in this study were regenerated in
vitro from scale leaves explants on Murashige and Skoog
(1962) medium supplemented with BA and NAA for which results have been published
elsewhere (Shushu et al., 2005). The bulbs were
originally obtained from market vendors of medicinal plants. The plants were
first transferred into commercial potting soil in 8 cm wide pots and acclimatized
in the green house for three months. After establishment into the soil, the
plants were transferred into larger pots in a mixture of field soil and commercial
potting soil (1:1). The plants were maintained in the green house and growth
and development of the plants were monitored.
Acclimatization of the plants to field conditions: In order to
assess the ability of the in vitro regenerated plants to grow in
natural field conditions, some of the plants were taken from the greenhouse
and planted outside in open plots and were not given any additional supplements
such as fertilizers. The plants were watered once a day only for the first
one month and thereafter left to grow under natural field conditions for
Determination of variants: During the growing period, the plants were
regularly checked for the presence of variant phenotypes in the green house
and the field and different phenotypes followed to see their stability in the
field. Morphological features were used to determine somaclonal variation (Bajaj,
1990; Tremblay et al., 1999). The number,
size of the fully expanded leaves, shape, colour and surface patterns of the
leaves and flowering was determined for each plant after one year of growth
in the field. The number of plants that survived and the growth habit of the
plants was also determined. Analysis of variance was used to determine differences
in the number and size of the leaves of variants.
Greenhouse plants: In vitro regenerated plants acclimatized
successfully when transferred in the soil in the green house with 90%
survival rate. The plants continued to grow very well in the green house
in semi-domesticated conditions (Fig. 1). They produced
large, attractive, shiny leaves and small inflorescences with purple flowers
(Fig. 1e, f). The plants produced
new leaves continuously when watered regularly. The plants also continued
to produce inflorescences successfully, some plants produced 2-3 inflorescences
at one time.
Acclimatization of the plants to field conditions: There was 100%
survival of plants transferred to field conditions. The plants were watered
for one month only after transfer. The plants survived periods of exposure
to very high temperatures (average temp. of 38°C) and drought. The
plants lost all leaves during winter season (May-July). Ninety six percent
of the plants resumed growth during rainy season (Oct.-April). The data
presented in Table 1 below was taken from new leaves
that developed during the warm wet period following the winter die-back.
||Ledebouria graminifolia plants: (a) Wild type
phenotype, (b) In vitro regenerated plants in the green house,
(c) Variants of in vitro regenerated Ledebouria in green house
(wild type, far left), (d) In vitro propagated plants new growth
after winter die off and, (e) Young inflorescence of regenerants and (f)
|| Variation in size and No. of leaves and flowering of
variants grown in the field
|Data are expressed as Mean±SE
Somaclonal variation: In vitro regenerated plants showed high
levels of variation in growth habit and leaf morphology in the greenhouse and
in the field (Fig. 1c, Fig. 2, Table
1). The field plants showed five visually distinct morphological types (Fig.
2). Morphotype 1 consisted of plants with large, fewer, bright green leaves
with small reddish speckles (Fig. 2 T1a and T1b).
This type showed a prostrate growth habit. Morphotype 2 consisted of plants
with large, lanceolate leaves which were slightly bright green (Fig.
2, T2). Morphotype 3 consisted of medium lanceolate, highly aristate
leaves (Fig. 2, T3). The leaves were grayish green
with rubbery appearance which curled inwards. Plants were slightly erect. Morphotype
4 had characteristics intermediate between 2 and 3 with grayish, slightly lanceolate
leaves (Fig. 2, T4). Morphotype 5 consisted of
very erect plants with curled, prominently linear leaves with rubbery appearance
and prominent reddish speckles at the base (Fig. 2, T5).
This type typically represented the parental phenotype of wild plants. There
was a significant difference in leaf length between the wild type phenotype
and all variants and also between Morphotypes 2 and 3. Leaf width also differed
between wild type and all variants but not among variants. There was also significant
difference in the number of leaves produced by the variants. All types showed
the prominent wavy, speckled leaves characteristic of the plant. Only Morphotype
1 and 2 produced flowers within the growing season.
|| Morphological variation of the in vitro propagated
Ledebouria sp. in the field
There was a difference in winter die back response among the variants.
Morphotype 1 was the first variant to lose the leaves, followed by Morphotype
2, 4, 5 and 3, respectively.
The present study has shown that in vitro regenerated indigenous bulb
Ledebouria graminifolia acclimatized very well both to greenhouse and
field conditions. The survival rate in the green house was 90%. Under a semi-domesticated
environment, Ledebouria produced bigger attractive leaves than wild plants
when provided with water and good growth medium such as a mixture of soil and
potting soil. Although the bulb produced small and not-so attractive inflorescences
(Fig. 1c, e). Ledebouria can
be grown as a potted ornamental plant for its large attractive speckled leaves,
features that are desirable in the industry. In vitro regenerated
Ledebouria plants showed remarkable tolerance and resilience to extreme
environmental conditions when grown in the field. The plants acclimatized well
after transfer to the field and exposed to natural conditions with minimum care.
There was 96% survival rate in the field. The plants went through a winter die
back (May-July) and were able to resume growth thereafter. Botswana has very
low levels of precipitation of between 200 and 600 mm per year and temperatures
can reach as high as 40°C. The ability of in vitro propagated plants
to withstand the dry conditions makes Ledebouria a suitable plant to
be used to fill an important gap in the flourishing and growing floriculture
industry in Botswana that is currently being dominated by exotic plants which
usually require high resource in-put, especially water. Ledebouria is
also an important medicinal bulb in Botswana hence extensively harvested from
the wild (Mutanyatta et al., 2003). Tissue culture
could be used to mass propagate the plant (Shushu et al.,
2005) for distribution to communities for use as an indoor or outdoor ornamental
plant as well hence contribute to the conservation of the plant.
Somaclonal variation: In vitro regenerated plants showed a wide
range of variation for leaf morphology such as leaf size, shape, colour and
growth habit in the greenhouse and in the field especially considering that
all these plants were regenerated from a single bulb. Somaclonal variation is
the variation observed among plants regenerated by tissue culture (Larkin
and Scowcroft, 1981). There a number of ways of assessing somaclonal variation,
variation in phenotype and response to environmental conditions was used to
assess variation in this study. Wild Ledebouria plants consist of linear
leaves with grey and dark green speckles. The leaf bases are decorated with
bright purple speckles. Morphological evaluation of the in vitro regenerated
plants after one year`s growth showed five distinct types of variations. Morphotype
1 had prostrate growth habit; had the least number but largest, attractive leaves.
This type however was not very well adapted to field conditions as shown by
early drying of leaves which started from the tips. Morphotype 2 had more leaves
than type 1. However, these were the only variants that produced flowers. Morphotypes
3 had semi-erect habit. This type produced more leaves that were hardy with
leathery appearance. These plants were more tolerant to drought than types 1
and 2 as they showed less signs of stress even under very hot and dry conditions.
The leaves of Morphotype 3 tended to curl inwards probably reducing the exposed
leaf lamina surface as an adaptation to drought. Morphotype 5 consisted of long,
linear leaves that curled inwards as well. As mentioned earlier, this represented
the phenotype naturally found in the wild. Morphotype 4 had characteristics
intermediate between 2 and 3 (Fig. 2). It was noted in this
study that variations in response to environmental conditions appeared to relate
to morphological and growth habit variation indicated above. That is, Morphotype
1 and 2 plants (with large, greener leaves and a prostrate habit) were prone
to desiccation more than other types. Leaf size could not have accounted for
this since there was no significant difference among variants except type 5.
The lack of tolerance to drought could have been due to the prostate growth
habit and fully exposed leaf lamina that were very close to the heated soil
which may have led to high exposed surface area for increased water loss. Morphotype
3, 4 and 5 were more tolerant to drought and heat; also they were the last plants
to lose leaves as winter season set in probably due to curling of leaves and
semi erect growth habit which reduced water loss from the leaves. Other factors
such as differences in leaf anatomical features may have been involved , this
aspect was not investigated in this study.
There are many factors possibly causing somaclonal variation. Genetic disorders
in the cells of initial explants, explant source, time in culture and growth
hormone have been cited to have an effect on the levels of somaclonal variation
(Karp, 1995). The variation seen in these plants could
have been due to the effects of growth substances. All the plants in this study
came from a single bulb as the source of explants and duration of the plants
in culture was 12 weeks. It is less likely that genotype and duration in culture
could have been the major source of the high variation seen in this study although
we can not completely discount it. Variation found in in vitro regenerated
plants may not be stable due to physiological disturbances and epigenetic influences
(Bajaj, 1990). The variation observed in these plants,
seemed to be stable variation as the phenotypes presented in this study were
obtained from second generation set of leaves that developed after a winter
die-back which was similar to phenotypes of plants that were transferred to
the field. Somaclonal variation has been found to be a useful alternative for
new product development. It offers an opportunity to uncover the natural variability
in plants especially for vegetative propagated plants (Larkin
and Snowcroft, 1981; Bajaj, 1990; Sahijram
et al., 2003). The high level of variation in phenotype unfolding
in this study was considered to be a good and valuable source of desired phenotypes
that can be commercially exploited in floriculture to meet the different consumer
market preferences in the industry.
The use of indigenous plants in the ornamental plant industry has really not
been given much attention although many of these plants can offer better alternative
to exotic species that are currently widely used in this industry. There are
very few countries in Africa that have directed effort towards popularizing
indigenous plants for ornamental use (Reinten and Coetzee,
2002) despite the growing interest in a green urban environment. Many of
the plants used for medicinal purposes can also be used as ornamental plants.
Research and commercial cultivation of indigenous bulbs for medicinal and ornamental
markets has made progress in other countries such as South Africa (McCartan
and Van Staden, 1999; Reinten and Coetzee, 2002) and
Turkey (Entwistle et al., 2002; Acar
et al., 2007). Botswana has a rich diversity of indigenous bulbs
such as Ledebouria sp. that are suitable for the ornamental plant industry
which could complement or offer ideal alternatives to exotic plants currently
dominating town landscapes and private house gardens. Ledebouria could
be mass propagated and distributed for planting therefore contributing to urban
biodiversity, sustainable utilization and conservation. Research efforts should
be directed towards efficient mass propagation methods, growth requirements
for improving their agronomic characteristics and markets.
This study is an extension of a research on exploitation of indigenous
geophytes in Botswana: Chemical and Biological investigations supported
by Research and Development Office of the University of Botswana, Research
vote No. R028, whose financial support is gratefully appreciated.
Acar, C., H. Acar and E. Eroglu, 2007. Evaluation of ornamental plant resources to urban biodiversity and cultural changing: A case study of residential landscapes in Trabzon city (Turkey). Build. Environ., 42: 218-229.
Direct Link |
Afolayan, A.J. and P.O. Adebola, 2004. In vitro propagation: A biotechnological tool capable of solving the problem of medicinal plants decimation on South Africa. AJB, 3: 683-687.
Direct Link |
Bajaj, Y.P.S., 1990. Somaclonal Variation- Origin, Induction, Cryopreservation, and Implications in Plant Breeding. In: Somaclonal Variation in Crop Improvement, Bajaj, Y.P.S. (Ed.). Springer-Verlag, Berlin, ISBN: 3 540 507 85-x.
Entwistle, A., S. Atay, A. Byfield and S. Oldfield, 2002. Alternatives for the bulb trade from Turkey: A case study of indigenous bulb propagation. Oryx, 36: 333-341.
Jain, S.M. and G.J. De Klerk, 1998. Somaclonal variation in breeding and propagation of ornamental crops. Plant Tissue Cult. Biotech., 4: 63-75.
Karp, A., 1995. Somaclonal variation as a tool for crop improvement. Euphytica, 85: 295-302.
Larkin, P.J. and W.R. Scowcroft , 1981. Somaclonal variation: A novel source of variability from cell culture for plant improvement. Theor. Applied Genet., 60: 197-214.
CrossRef | Direct Link |
Mander, M., J. Mander and C. Breen, 1996. Promoting the Cultivation of Indigenous Plants for Markets: Experience from Kwazulu-Natal, South Africa. In: Domestication and Commercialization of Non-Timber Forest Products in Agroforestry Systems, Van Honten, H. and K. Kebaara (Eds.). FAO Technical Publication, Italy, Rome, ISBN: 9251037019.
Mateke, S. and T. Matlhare, 2000. Under-utilized plants- their conservation and sustainable use by rural communities in arid, semiarid ecosystems of Botswana. Proceedings of the 2nd Botswana Workshop on Plant Genetic Resources: Plant Genetic Resources- A Heritage for the Future, June 20-23, 2000, Printing and Publishing House, Gaborone, Botswana, pp: 18-31.
Mc Cartan, S.A. and J. Van Staden, 1999. Micropropagation of members of Hyacinthaceae with medicinal and ornamental potential. Rev. S. Afr. J. Bot., 65: 361-369.
Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum, 15: 473-497.
CrossRef | Direct Link |
Mutanyatta, J., Matapa, B.G. Shushu and D.D. Abegaz, 2003. Homoisoflavonoids and xanthones from the bulbs of wild and in vitro regenerated Ledebouria graminifolia and cytotoxic activities of some of the homoisoflavonids. Phytochemistry, 62: 797-804.
Direct Link |
Podwyszynska, M., 2005. Somaclonal variation in micropropagated tulips based on phenotype observation. J. Fruit Ornam. Plant Res., 13: 109-122.
Direct Link |
Reinten, E. and J.H. Coetzee, 2002. Commercialization of South African Indigenous Crops: Aspects of Research and Cultivation of Products. In: Trends in New Crops and New Uses, Janick, J. and A. Whipkey (Eds.). ASHS Press, Alexandria, ISBN: 097075655.
Rositter, S., S. Pellegrin, K. Hill, A. Bunting, S. Robinson, T. Mason, T. Boitumelo and A. Madiba, 1997. The Present Importance and Future Potential of Veld Products in the Livelihoods of Villagers from the Tswapong Hills. 1st Edn., Botswana Society, Gaborone.
Rout, G.R., A. Mohapatra and S.M. Jain, 2006. Tissue culture of ornamental pot plant: A critical review on present scenario and future prospects. Biotech. Adv., 24: 531-556.
Direct Link |
Sahijram, L., J.R. Soneji and K.T. Bollamma, 2003. Analyzing somaclonal variation in micropropagated bananas (Musa spp.). In vitro Cell. Dev. Biol. Plant, 39: 551-556.
CrossRef | Direct Link |
Scowcroft, W.R. and P.J. Larkin, 1988. Somaclonal Variation. In: Applications of Plant Cell and Tissue Culture, Bock, G. and L. March (Eds.). CIBA Foundation Symp. Wiley, Chichester.
Shushu, D.D., J. Mutanyatta and B.M. Abegaz, 2005. In vitro propagation and phytochemical investigation of Ledebouria graminifolia, an important medicinal plant in southern Africa. J. Herbs Spices Medicinal Plants, 11: 97-107.
Direct Link |
Taylor, F.W., 1985. The Potential for Utilization of Indigenous Plants in Botswana. In: Plants for Arid Lands, Wickens, G.E., J.R. Gooding and D.V. Field (Eds.). George Allen and Unwin, London.
Tremblay, L., C. Levasseur and F.M. Tremblay, 1999. Frequency of somaclonal variation in plants of black spruce (Picea mariana, Pinaceae) and white spruce (P.glauca, Pinaceae) derived from somatic embryogenesis and identification of some factors in genetic instability. Amer. J. Bot., 86: 1373-1381.