Protocol Establishment for Micro propagation and in vitro Callus Regeneration of Maulavi Kachu (Xanthosoma sagittifolium L Schott.) From Cormel Axillary Bud Meristem
The effects of various growth regulators on organogenesis from cormel axillary bud meristems of cultivated species of Maulavi kachu (Xanthosoma sagittifolium (L). Schott. were investigated. Highest percentage (65%) direct regeneration takes place within 25 days of culture but significant number of shoot per plant and length of shoot did not found significantly. Highest percentage (90%) calluses were initiated from axillary buds on MS medium supplemented with 1 mg L-1 BAP+2.0 mg L-1 NAA. Light green, greenish friable or semi friable calluses were found. The highest fresh weight of callus was 11.60 g. These calluses were cultured into MS media containing with 3 mg L -1 Kn + 2 mg L-1 NAA to induce plantlets regeneration. Highest percentage (86%) regeneration was observed with single or aggregated green shoot buds. The average number of green shoot, daughter buds and plantlets were 2.60 and average length of shoots were 7.0 cm with the same medium. Highest percentage of root induction and proliferation were also found into media containing 0.4 mg L-1 IAA and number of roots and root length of regenerated plantlets were 7.90 No. and 6.90 cm, respectively. Plantlets were then established with normal and natural fertile soil on plastic pot showed best performance in acclimatization.
Maulavi kachu is one of most important root and tuber crop world wide (Jennings,
1987; Onwueme and Charles, 1994) and cultivated in
the tropics and subtropics as important root crops and are widely affected by
dasheen mosaic viruses (Zettler et al., 1970).
It is a neglected edible aroid crop in Bangladesh, mainly grown by small scale
farmers and attempts to improvement have therefore been limited. Xanthosoma
sagittifolium (L.) Schott commonly known as maulavi kachu in Bangladesh,
belongs to the monocotyledonous family Araceae. The corm, cormels and green
leaves of maulavi kachu are important source of carbohydrates for human nutrition,
animal feed (Ndoumou et al., 1995; Nyochembeng
and Garton, 1998) and of cash income for village farmers (Tambong, 1997).
The crop is mainly cultivated by small scale farmers (Onwume
and Charles, 1994) in Asia, Africa and Latin America (Wilson,
1984). In spite of its importance as a staple food in many countries, maulavikachu
could receive very little research attention (Goenaga and
Heperly, 1990) and is regarded as an under exploited and insufficiently
studied crop (Nguyen and Nguyen, 1987; Giacometti
and Leon, 1994; Watanabe, 2002).
In recent years biotechnology offers enormous opportunity for improvement of
food crops which equally hold the potential for the improvement of aroids including
maulavikachu (Xanthosoma sagittifolium (L.) Schott) (Kacker
et al., 1993). Previous study on callus initiation in Xanthosoma
sp. using shoot and/or meristem tips has been reported (Hartman,
1974; Strauss et al., 1979; Licha
et al., 1982. Acheampong and Henshaw (1984) also reported development
of protocorms directly from shoot tips in agitated media. Many researchers already
reported some tissue culture research study on some species of aroids. Tissue
culture techniques open up many possibilities for sustainable production and
improvement of crops. Meristem culture of maulavi kachu have been reported (Tsala
et al., 1996; Zok et al., 1998) and
meristem derived plants perform better in terms of yield than virus infected
in vitro plants (Reys et al., 2006). Meristem derived plants are
also important for a safe exchange of germplasm between countries. Tissue culture
also gives the possibility to generate new genotypes through somaclonal variation
and a variety of morphological changes in callus derived maulavi kachu (Xanthosoma
sagittifolium (L.) Schott) plants has been reported by Gupta
(1985). Plant regeneration from tissues and cell culture is important in
achieving rapid clonal multiplication, recovery of pathogen free plants, preservation
of valuable germplasm and induction of chromosomal and genic variation (Murashige,
1974; Vasil and Vasil, 1980; Larkin
and Scoweroft, 1981). The technique is particularly valuable in vegetatively
propagated plants,where stem segments and tubers encourage the spread of many
pathogens resulting in severe loss of plant vigor and productivity like maulavi
kachu (Xanthosoma sagittifolium (L.) Schott). In this study we describe
a Reyscomplete in vitro protocol for Micro propagation, callus induction
and their regeneration for maulavikachu offering the scopes for culturing virus
free propagules and under taking genetic engineering research of this crop for
its further improvement.
MATERIALS AND METHODS
In the present study axillary bud meristem used as explants of Maulavikachu
were collected from field grown (2004-2006) cultivated species from experimental
research farm of Institute of Biological Sciences, Rajshahi University. Bangladesh.
Explants and Media Preparation
The collected axillary bud meristem attached with cormels washed under running
tap water. Then terminal sprouts were excised from cormels and surface sterilized
with 1-3 drops of tween-80 and Savlon per 100 mL water for 15 min followed by
several washing with sterilized distilled water.Surface sterilization was carried
by dipping 0.10% HgCl2 by gentle shaking for 15 min. The sterilized
materials were washed 4-5 times with sterile distilled water immediately to
remove all traces of HgCl2. The basic culture medium was MS (Murashige
and Skoog, 1962) supplemented with various concentrations and combinations
of IAA, 2, 4-D NAA, BAP and Kn and. The media were adjusted to pH 5.8 and subsequently
were jelled with 0.8% agar and 3% sugar and autoclaved for 20 min at 121°C
and 1.1 kg cm2. Explants were inoculated longitudinally on to media
containing culture bottle in a laminar flow cabinet. Cultures were incubated
at 26+1°C under the warm in fluorescence light intensity varied from 2000-3000
lux. The photo period was maintained 16 h light and 8 h dark. Calluses were
aseptically weighed and regenerated shoot lengths were measured.
RESULTS AND DISCUSSION
For direct regeneration three auxins (NAA, IAA, 2, 4-D) and Cytokinins (BAP
and Kn) were used in MS basal medium in different concentration s and combinations
and their data are presented in Table 1. Highest percentage
(65%) of plantlets regeneration was found with the number of shoot per plant
(1.00) and shoot length (0.783 cm) in MS media supplemented with 0.50 mg L-1
BAP+1.0 mg L-1 IAA followed by media containing 0.50+1.50
mg L-1 with same number of shoot and shoot length.
||Effect of different concentration and combination of BAP,
KIN, IBA, NAA, 2, 4-D on direct shoot regeneration from axillary cormel
bud explants of Maulavi kachu (Xanthosoma sagittifolium (L.) Schott.
and Data were recorded after 4 weeks of culture
||Showing different stages of direct regeneration in Xanthosoma
sagittifolium (L.) Schott, (a) Insertion of fresh axillary cormel bud
into the MS medium supplemented with 0.5 mg BAP L-1 + 1.0 mg
IAA L-1, (b) Shoot regeneration after 20 days from insertion
to the medium containing with 0.5 mg BAP L-1 +1.0 mg IAA L-1,
(c) Shoot regeneration after 25 days from insertion to the medium with same
medium and (d) After 30 days observation
In most observation the and regeneration percentage is slow and low. Data was
recorded Four weeks after from culturing date (Table 1, Fig.
1). Reys (2006) acheived the highest percentage of
regenerating explants from NG,cocoyam (83%) using 1.0 mg L-1 IAA
in combination with 0.5 mg L-1 BAP to the medium. Faria
and Illg (1995) reported that the addition of 10 μM BA along with 5
μM indole-3 acetic acid (IAA) or 5 μM NAA induces a high rate of shoot
proliferation of Zingiber spectabile. They also noticed that the number
of shoots/explant depends on concentration of the growth regulators and the
particular genotype. Plant regeneration was obtained by (Tsala
et al., 1996) from shoot tip explants cultured in MS medium devoid
of any Phythormone after twenty days.
The basal media only MS (Murashige and Skoog,1962)
was used in callus initiation. Shoot tip explants were cultured on MS media
supplemented with different concentrations and combinations of 2,4-D, Kn, BAP,
NAA and IAA. Data were recorded at the induction of the calluses,cultures were
maintained under 16 h light and 8 h dark regime and results obtained (Table
2 and Fig. 2). Callus proliferation was not noticed in
all media formulations.
||Effect of phytohormones on induction of calluses from axillary
bud explants of Maulavi kachu (Xanthosoma sagittifolium L. Schott)
|-indicates no response, * Indicate poor growth, ** Indicates
moderate growth, *** Indicates profuse growth. - No. of root /shoot growth,
+ root/shoot (1-3)/callus, LG = Light Green, DG = Dark Green, C = Compact,
F = Friable
||Different stages of Callus induction in Xanthosoma sagittifolium
(L.) Schott, (a) Insertion of axillary bud to medium supplementing 1 mg
L-1 BAP+2.0 mgL-1NAA, (b) Advance stage of Callus
proliferation media containing with 1mg L-1 BAP+ 2.0 mg L-1
NAA (c) and Callus proliferation after 70 days, Callus formation after 85
days observation Ms media supplemented with supplemented with 1 mg L-1
BAP+ 2.0 mg L-1 NAA
But there was a wide variations in morphological maturity and percentage of
callus formation among them. Callus initiation occurred within 70-80 days depending
upon the concentration and combination of hormones. Frequency of callus formation
ranged from 15-90%. Highest percentage (90%) of callus formation occurred in
MS medium containing 1 mg L-1 BAP+2 mg L-1 NAA (Fig.
2) and followed by 85% in MS medium containing 3 mg L-1 Kn +2
mg L-1 NAA. The lowest percentage of callus (15%) formation was observed
in media having 1 mg L-1NAA. In most cases calli were green, light
green and dark green and texture of calluses were compact and friable. The optimum
callus growth in terms of fresh weight was 11.60 g. in 1 mg L-1 BAP+2
mg L-1 NAA and followed by 10.40 g in 3 mg L-1 BAP+0.5
mg L-1NAA. The minimum weight was 3.30 g in 3 mg L-1 NAA.
Most of calluses were responded with shoot positively and some calluses showed
adventive root formation. Tsala et al. (1996)
also showed that in vitro plantlets were obtained when shoot apices were cultured
in MS medium supplemented with 10-5 M 2 mg L-1 Kn, Stem
apices of Xanthosoma sagittifolium developed fast growing calluses and
were transferred to medium without growth regulators after 12-16 weeks (Gomez
et al., 1992). NAA and 10-6 Kn M.MS medium supplemented
with 5 mg L-1 NAA, 100 mL-1 coconut milk and 2 mg L-1
Kn produced compact green calluses and organogenesis occurred within eight weeks
(Gupta, 1985). After maintenance for eight weeks in
MS medium supplemented with 25 mg L-1 IAA and Nyochembeng LM and
Garton S (1988) reported calluses were initiated from shoot tips and petiole
explants and proliferated on medium containing 1.36 μM dicamba. He also
reported that thidiazuron (0.045 μM) enhance callus production when dicamba
(13.5 μM) was used and was more favourable to petioles that shoot tips.
Calli derived from the cormel bud meristematic tissues were transferred
to cytokinin enriched media for organogenesis. For shoot differentiation light
green compact calli were sub cultured on MS medium supplemented with different
combinations and combinations of BAP and Kn alone and in combination with different
concentration of NAA, 2, 4-D, IBA and IAA.Morphogenic potentialities of cultured
calli varied with hormonal treatments (Table 3 and Fig.
3). Calli sub cultured with different concentration of IAA, BAP, Kn alone
failed to differentiate any callus shown (data not shown). Calli produced shoots
only when NAA was combined with cytokinins.
||Effect of phytohormones on regeneration of calluses from axillary
bud explants of Malavi kachu (Xanthosoma sagittifolium L.schott)
|-: Indicates no response,*Indicate poor growth,** Indicates
moderate growth, *** Indicates profuse growth. No. of root /shoot
growth, + root/shoot (1-3)/callus, LG = Light Green, DG = Dark Green, C
= Compact, F = Friable
||Showing different stages of callus regeneration of Xanthosoma
sagittifolium (L.) Schott, (a) Callus insertion to the medium supplementing
with 3 mg Kn L-1 +2 mg L-1 NAA, (b) Proliferation
or regeneration of callus growing shoots with same medium after 25 days
of Culturing, (c) Multiple shoot production after 30 days and (d) Multiple
plantlets produce after 30-40 days with same medium
Highest percentage (89%) shoot regeneration was recorded in 1 mg L-1 BAP+2.0
mg L-1 NAA (Fig. 2) and followed by 86% in media
having 3 mg L-1 Kn+2.0 mg L-1 NAA. Lowest percentage (36.00%)
Callus regeneration was observed in media with having 3 mg L-1 Kn+3
mg L-1 NAA. The highest percentage of shoot per callus was recorded
2.60 in media having 1 mg L-1 BAP+2.0 mg L-1 NAA. The
lowest number of shoots 1.20 per callus was recorded in media having 1 mg L-1
BAP+1 mg L-1 NAA and 3 mg L-1 Kn+3 mg L-1 NAA.
Highest length of shoots 7.00 cm was recorded in 1 mg L-1 BAP+2 mg
L-1 NAA and followed by 3.20 cm in media containing 1 mg L-1
BAP+1.5 mg L-1 NAA and the lowest length of shoot was 1.30 cm recorded
in 3 mg L-1 BAP+3 mg L-1 NAA. Fresh weight of callus with
shoot was highest in media having 1 mg L-1 BAP+2.0 mg L-1
NAA followed by 12.00 g. in media supplemented with 3.0 mg L-1 Kn+2.0
mg L-1 NAA and the lowest weight (5.30 g) was recovered in media
having 1 mg L-1 BAP+1 mg L-1 NAA. Nyochembeng and Garton
(1988) also reported that callus regeneration or producing single or aggregated
shoot buds sub cultured into media containing 0,0.049 and 0.49 μM 2 iso
|| Root induction, Ms medium supplementing with 0.4 mg L-1
||Effect of different Concentration of Auxin on root induction
from the axillary bud explant derived plants of Maulavi kachu (Xanthosoma
sagittifolium L. Schott)
|- = Not response
Induction of callus and subsequent organogenesis were by a two step system
for plant regeneration. By the manipulation of growth regulating substances,
it was possible to regenerate functional plants in a one step system. Plantlets
possessed well develop shoot and root systems were regenerated from the excised
meristems within eight weeks of culturing.
Plants free from specific pathogens, through tissue culture techniques have
been reported in various vegetatively propagated crops e.g., Potato, Sugercane
and asparagus (Murashiege and Skog, 1978). In the present
studies dasheen mosaic virus and other pathogen free plants were regenerated
from the meristematic domes of Xanthosoma sagittifolium (L.) schott and
subsequently the regenerants were showed good performance in growth on natural
growing soil medium.
Regenerated plants were then cultured on to a rooting medium containing
different concentration and combination of growth regulators of which only 0.4
mg L-1 IAA showed best performance for root induction and profuse
root proliferation occurred within 7-10 days from culture (Table
4 and Fig. 4).
Plantlets with rooting were removed from the culture vessels and then washed
with running distilled water or running tap water and then transferred to wet
fertile loamy soil contained plastic pots for hardening. Within one months or
before the plantlets showed best performance for acclimatization.
This work was supported by UGC (University Grants Commission of Bangladesh) and nominated the author for financial support as fellowship for this research. The author appreciated thanks to UGC authority for financial help for this research.
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