In vitro Shoot Tip Culture of Sugar-cane (Saccharum officinarum) Variety Isd 28
Multiple shoots were obtained from shoot tip explant of sugar-cane (Saccharum officinarum) cultured on MS medium supplemented with BAP (0.5-2.0 mg l-1), Kn (0.1-0.5 mg l-1) and IBA (0.1-0.5 mg l-1). Roots were induced in in vitro regenerated shoots on half MS medium supplemented with 5.0 mg l-1 NAA, IBA and IAA. Plant regeneration from shoot tip was the highest on MS medium supplemented with BAP 2.0 mg l-1 and IBA 0.5 mg l-1. The reported experimental findings present a method of plant regeneration of sugar-cane variety Isd 28 through shoot tip culture. The plantlets were successfully transferred to soil and the percentage of survivability under ex vitro condition was 70.
Sugar-cane is a member of the genus Saccharum from family Gramineae.
Varieties of sugar-cane are highly heterogeneous and generally multiplied vegetatively
by stem cutting. Time required and continuous contamination by systemic diseases
are the serious problems to multiply an elite genotype of sugar-cane in the
open field (Nand and Singh, 1994). Tissue culture of sugar-cane has received
considerable research attention because of its economic importance as a cash
crop. Plant regeneration through tissue culture technique would be a viable
alternative for improving the quality and production of sugar-cane. Initial
attempts to regenerate plants through in vitro technique were made on
sugar-cane by Nickell (1964) and Heinz and Mee (1969). Protocols for in vitro
plant regeneration of sugar-cane through callus culture, axillary bud and
shoot tip culture have been developed by many authors (Barba et al.,
1978; Sauvaire and Glazy, 1978; Heinz et al., 1977; Lee, 1987). Induction
of callus and regeneration of plants using sugar-cane varieties of Bangladesh
were reported elsewhere (Islam et al., 1982; Hossain et al., 1993;
Karim et al., 2002). However, reports are scarce on shoot tip culture
in sugar-cane varieties of Bangladesh. Multiplication and germplasm preservation
of sugar-cane is possible and for this purpose shoot tip has a greater potentiality.
Therefore, the present investigation has been undertaken to establish plant
regeneration protocol through shoot tip culture in sugar-cane using variety
Baksha et al.: In vitro shoot tip culture of sugar-cane
Materials and Methods
The experiment was conducted at Biotechnology Laboratory in Bangladesh Sugar-cane
Research Institute (BSRI), Ishurdi, Pabna, Bangladesh during the period of 2000-2001.
The plants of sugar- cane variety Isd 28 developed by BSRI, Bangladesh were
used as experimental material. Shoot tips were collected from juvenile sugar-cane
plants (3-4 months age) and were used as explants. Sterilization of explants
was carried out using 0.1% HgCl2 after washing thoroughly under tap water for
7-10 min. Subsequently the explants were washed gently with sterile DDH2O (double
distilled water) in aseptic condition under laminar flow hood. Shoot tips of
2-4 mm were excised and placed on MS (Murashige and Skoog, 1962) medium supplemented
with different combinations of auxin and cytokinin to identify the appropriate
media combinations for regeneration of sugar-cane through shoot tip culture.
Media were consisted of 3% sucrose, 0.6% agar, pH was adjusted to 5.7 before
addition of agar and autoclaved at 120°C for 15 min. Explants were incubated
at 25±2°C under 16 h photoperiod regime. The exp eriments were replicated
two times and the means and standard errors of the results were calculated.
Results and Discussion
In experiment reported here, different concentrations and combinations of auxin
and cytokinin were used in MS medium for multiple shoot regeneration from shoot
tip of sugar-cane. Yutaka et al. (1998) reported that combinations of
phytohormones often determine the course of morphogenesis e.g. shoot organogenesis
or embryogenesis. For multiple shoot regeneration, shoot tips were remarkably
influenced by types and concentrations of the auxins and cytokinins used. The
cytokinin BAP was more effective than Kn and IBA for shoot formation. Low auxin
and high cytokinin supplementation in medium favoured the induction of multiple
shoot regeneration. Various combinations of BAP with IBA or Kn were tried. The
maximum response for multiple shoot initiation were found when explants were
cultured on MS medium supplemented with 2.0 mg l-1 BAP + 0.5 mg l-1
IBA, 1.0 mg l-1 BAP + 0.5 mg l-1 IBA and 1.0 mg l-1
+ 0.5 mg l-1 Kn. On these media 70-75% explants produced 2-6 shoots
from a single shoot tips within 2-3 weeks (Fig. 1A-C). Skirvin
(1984) has revealed the effects of different hormone combinations on proliferation
and elongation. Number of explants cultured and their responses to shoot regeneration
and growth were recorded (Table 1). The highest shoot length
was 4.5±0.01 cm followed by 4±0.35 and 4±0.21 cm. The regenerated
shoots were multiplied manifolds when they were sub-cultured in the same medium
within three weeks.
The regenerated shoots were devoid of roots. So, for root induction the shoots
were excised separately and placed on rooting media. The same concentration
(5 mg l-1) of IAA, NAA or IBA were used alone in half MS medium for
healthy and profuse root induction. Rooting of shoots may be affected by pH,
auxin level and nutrient concentration of the root induction media (Dannis and
James, 1993). Best response was observed when 5 mg l-1 of NAA was
used in half MS medium (Table 2 and Fig. 1D).
Root developed in IAA or IBA containing medium was poor in quality. These results
agree well with the previous findings of Nadar and Heinz (1977), who reported
that preferred auxin for root initiation was NAA.
|| Effects of auxin and cytokinin in MS medium on shoot regeneration
from shoot tips of Sugar-cane variety Isd 28. There were 18-20 explants
for each treatment and data (X ± SE) were recorded after 2-3 weeks
|6- Benzylaminopurine (BAP), Kinetin (Kn), Indole-3- butyric
acid (IBA)*Mean value.**Standard error.
|| Effects of different levels of IBA, NAA and IAA on root formation
in Sugar-cane variety Isd 28. There were 18-20 explants for each treatment
and data (X ± SE) were recorded after 2-3 weeks of culture
|Indole-3- acetic acid (IAA),1-naphthaleneacetic acid (NAA),
Indole-3- butyric acid(IBA)
*Mean Value.**Standard Error.
The proper stage of root development was another criterion for selecting plantlets
to be transferred to the soil. In vitro regenerated plantlets were transferred
to small pots containing mixture of soil and sand (2:1) for future establishment
(Fig. 1E). In general it has been reported that plants regenerated
from meristem (shoot tips) are very similar both phenotypically and genotypically
to the mother plants (Amato, 1977).
|| Different stages of in vitro derived regenerated plantlets from
shoot tip culture of sugar-cane.
||Initiation of axillary shoots on MS medium with 1.0 mg l-1 BAP + 0.5 mg
||Development and multiple shoot formation on MS medium with 2.0 mg l-1
BAP + 0.5 mg l-1 IBA after two-three weeks of culture.
||Root formation on half strength MS medium with 5.0 mg l-1 NAA after four
weeks of culture.
||In vitro raised plantlets into soil in polythene bags.
Grisham and Bourg (1989) compared two micro propagation methods for the cultivars
CP65-357 and CP70-321 and found that shoot tip culture was better than leaf
roll culture for plant production. The rapid regeneration and germplasm preservation
of elite sugar-cane variety is possible and for this purpose shoot tip has greater
potential towards the multiple shoot regeneration. Present report showed that
among the media, MS medium containing 2.0 mg l-1 BAP+0.5 mg l-1 IBA, 1.0 mg
l-1 BAP+ 0.5 mg l-1 IBA and 1.0 mg l-1 BAP + 0.5 mg l-1 Kn was more effective
for multiple shoot formation. Furthermore, it has been demonstrated that for
shoot regeneration the combination of auxin and cytokinin was essential. Sugar-cane
variety Isd 28 displayed an excellent regeneration capacity. This study of micro
propagation has given a rapid technology compared with conventional technique
for multiplication and germplasm preservation of elite sugar-cane varieties.
Therefore, shoot tip culture offers a definite scope for further improvement
of this well adapted genotype through gene manipulation using other biotechnological
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