In vitro Mass Propagation of Sugarcane (Saccharum officinarum L.) var. Isd 32 through Shoot tips and Folded Leaves Culture
Plant tissue culture is the most commercially successful aspect of plant biotechnology, which has introduced an exciting new phase into plant propagation and breeding. This paper presents a research finding that establish an efficient regeneration method for mass propagation of sugarcane var. Isd 32 using shoot tip and folded leaf as explants. In this study, in vitro shoot tip and folded leaf culture techniques were used for mass propagation and quality production of sugarcane plantlets. The explants of sugarcane var. Isd 32 was cultured on MS + 1.5 mg L-1 BA + 0.5 mg L-1 NAA for shoot tip and MS + 0.5 mg L-1 Kin + 5.0 mg L-1 NAA for folded leaf. Culture after 30 days, about 90 and 80% of the shoot tip and folded leaf explants, respectively were successfully regenerated shoots. The average number and length of shoots from shoot tip explants was observed as 17.20±1.30 and 7.20±1.40 cm, respectively. Similarly, the average number and length of shoots from folded leaf explants was observed as 20.20±2.20 and 6.70±1.20 cm, respectively. Presence of callus and morphological variants were not observed during the passage of in vitro culture. There is no efficient regeneration technique that had been established for mass propagation of sugarcane var. Isd 32. The proposed technique can be used to enhance mass production of sugarcane crop economically especially with the present trend of demand of sugarcane in the region.
Sugarcane is one of the important cash crops in Bangladesh. It is the main source of sugar. It is a perennial herb and belongs to the family Gramineae. It is mainly propagated vegetatively by three budded setts. This traditional practice can result high production cost and also of large amount of sugarcane involvement. Tissue culture materials could be an alternative to overcome those variabilities and to produce uniform propagules for the cultivation. Recently, a large number of identical clones by in vitro culture techniques were routinely used for a wide range of plant species (Biondi, 1986). In recent decades, in vitro shoot tip culture for mass propagation of sugarcane was reported by many authors (Hendre et al., 1983; Lee, 1987; Nand and Singh, 1994). In vitro regeneration through leaf sheath culture was developed (Samad and Begum, 2000) and plant regeneration through callus culture was also established (Nickell, 1977). But, the nutritional requirements for in vitro culture vary according to genotypes as well as explant used. It is documented that commercial propagation of sugarcane through setts is slow and pathogen keep on accumulating generation after generation, which reduces the yield and quality of sugarcane. Therefore, efficient regeneration method is needed for mass propagation of this crop, which provide uniform and disease free propagules for potential and quality production of sugarcane. Thus, the study was conducted to develop an efficient protocol for the large scale in vitro regeneration of an elite variety Isd. 32 using shoot tip and folded leave as explants.
MATERIALS AND METHODS
Shoots containing shoot tips and folded leaves of a mature plant of sugarcane
var. Isd 32 were collected from the field of Atomic Energy Research Establishment,
Savar Dhaka. Shoots were washed thoroughly using household detergent, Trix for
15 min under running tap water. Subsequent sterilization was carried out in
the laminar air flow cabinet under aseptic conditions. Shoots were sterilized
in 0.1% mercuric chloride for 5 min. Rinsing was done 3 times with sterile distilled
water. The shoot tips and folded leaves (Murashige and Skoog, 1962) were exposed
after excision and inoculated into MS media supplemented with different concentrations
of BA and Kin alone or in combination with NAA for shoot induction. Subcultures
were done 15 days interval to promote multiple shoots and healthy plantlets
formation. Morphologically healthy shoots were excised individually and transferred
to half strength of MS media supplemented with different concentrations of IBA,
IAA and NAA for root induction. The sucrose concentration was used 30 g L-1
and the pH of the media adjusted to 5.8 prior to autoclaving. Cultures were
incubated at 26±2°C with a 16 h illumination of 21.8 μmol cm-2
sec-1 provided by cool white fluorescent tubes. The experiment was
conducted with 30 explants per media combination. Data were recorded 30 days
after inoculation. Observations on cultures were carried out every alternate
day. The study was conducted on February 2006 in the laboratory of Plant Biotechnology
and Genetic Engineering Division, Institute of Food and Radiation Biology, Atomic
Energy Research Establishment, Savar, Dhaka. The variety was collected from
the Sugarcane Research Institute, Ishurdi, Bangladesh and planted in the field
of Atomic Energy Research Establishment, Savar, Dhaka.
RESULTS AND DISCUSSION
Enlargement, swelling, browning and unfolding of explants were observed culture
after 7 days at all media combinations investigated. Cut ends of the folded
leaf explants became swollen and started to produce tiny meristems after 15
to 20 days of culture. In shoot tip explants, auxiliary shoot proliferation
occurred from nodes where leaf base adjacent to the stem. But adventitious shoot
proliferation was observed from cut ends and mid ribs of folded leaf explants.
Shoot proliferation from shoot tip and folded leaf explants differed according
to concentrations of cytokinin alone or the combinations of cytokinin and auxin
used (Table 1). Among the cytokinins used, BA was found to
be the best for shoot proliferation from shoot tip explants and 1.5 mg L-1
BA was found most optimum, in which 70% explants produced shoots. The average
number of shoot produced per explants was 8.30±0.60 and the average shoot
length of 6.50±0.80 cm were observed in this medium. On the other hand,
explants of folded leaf did not response any in such media of cytokinin alone
This is obvious from the result that single hormone has no effect for in
vitro regeneration from folded leaf explants of sugarcane cultivar. Therefore,
the study suggests that combination of cytokinin and auxin is necessary for
in vitro proliferation of folded leaf explants in Saccharum sp.
The superiority of BA over Kin was also found among the different combinations
used in shoot tip explants. Among the combinations, 1.5 mg L-1 BA
+ 0.5 mg L-1 NAA was found to be best, in which 90% of the shoot
tip explants induced shoots. The number of shoot produced per explant was 17.20±1.30
and the average shoot length of 7.20±1.40 cm were observed in this medium
||Effects of BA, Kin, BA + NAA and Kin +NAA on the in vitro
shoot induction of sugarcane var. Isd 32 using shoot tip and folded leaf
as explants at 30 days
|- = no response from the culture, SE = Standard Error
||In vitro plant regeneration through shoot tip and folded
leaf culture (a) multiple shoot formation from shoot tip on MS + 1.5 mg
L-1 BA + 0.5 mg L-1 NAA (b) multiple shoot formation
from folded leaf segment on MS + 0.5 mg L-1 Kin + 5.0 mg L-1
NAA (c) root induction from the shoots and (d) in vitro raised plant
in earthen pot
Positive effects of BA in regeneration of Saccharum sp. using meristem
tissue as explants were reported by several authors (Paul et al., 1993;
Tripathi et al., 2000). On the other hand, folded leaf explants was observed
to be the best on the media combination of 0.5 mg L-1 Kin + 5.0 mg
L-1 NAA, in which 80% explants induced shoots (Fig.
1b). The average number of shoot induced per explants was 20.10±2.50
and the average shoot length of 6.20±1.50 cm were observed in this medium.
These results are in agreement with the previous study of sugarcane shoot tip
culture in liquid modified medium of MS + 0.25 mg L-1 BA + 0.25 mg
L-1 Kin as reported (Nand and Singh, 1994). These results are also
similar to those obtained in auxiliary bud culture of sugarcane cultivars using
the combinations of BA and Kin (Paul et al., 1993; Gupta and Bhatia,
2004; Sreenivasan and Sreenivasan, 1985). Good responses towards shoot formation
and shoot multiplication from meristematic tissue of Saccharum sp. using
the combination of BA and Kin was also reported (Hendre et al., 1983).
In comparison to previous study, this study involved only 1 media combination
for shoot development and multiplication. The study also involved to identify
media combinations for maximum shoot proliferation with the different variety
Isd 32. It is an evident in this study that folded leaf explants did not response
for shooting in the media containing BA or Kin alone and shoot tip explants
did not response in the media combination containing 3 mg L-1 or
more than 3 mg L-1 NAA. These might be due to imbalance media composition
used or due to the metabolism of toxic by-products such as polyphenol oxidases
produced by the plant tissue. Tripathi et al. (2000) reported use of
BA promoted callusing tendency of the explants of cv. Cose 95436, but this study
did not show any callusing tendency of the explants during the passage of in
||Effects of IBA, IAA and NAA on half strength of MS media in
root induction from in vitro raised shoots of sugarcane var. Isd
32 at 30 days
|SE = Standard Error
On the other hand, Sood et al. (2006) regenerated plants using IAA,
BA and GA3 in cv. CoJ 64. Therefore, difficulties of regeneration
and non reproducibility of results might be due to the effect of genotypes.
The rooting response differed according to concentration of different auxins
used (Table 2). Among the auxins used, NAA was found to be
the most responsive for root induction and 2.5 mg L-1 NAA was found
most optimum, in which 90% shoots rooted within 15 days of culture. The average
number of root induced per shoot was 18.40±2.10 and the average root
length of 7.45±1.25 cm were observed in this medium (Fig.
1c). These observations were closely related to the previous study on root
induction of Sugarcane cultivars (Nand and Singh, 1994; Paul et al.,
1993) and also in Eucalyptus citriodora (Gupta et al., 1981) as
using NAA as stimulant. The importance of NAA by successful induction of roots
of sugarcane plants in vitro was also described (Anonymous, 1982; Sood
et al., 2006). The previous study also showed that the use of IBA or
IAA was not very effective for sugarcane root induction in vitro. However,
comparatively healthy rooted shoots were taken out from the culture vessels
and washed gently under running tap water to get rid of agar. The in vitro
rooted plantlets were then transferred to earthen pots (Fig. 1d)
containing a mixture of soil and compost (2:1) and covered with transparent
polyethylene lid to maintained high humidity. The polyethylene lids were removed
after 7 days. The plantlets were kept in a shade and misted twice a day. About
90% of the plantlets were resumed new growth within 30 days and were transferred
to the field.
Out of 50 in vitro raised plants, 45 grew to out door environment. The protocol described in this study is reproducible and long term high frequency in vitro regeneration of Sugarcane var. Isd 32. Therefore, this protocol might be use for in vitro propagation of a wide range of sugarcane cultivars.
All the existing cultivars of sugarcane are susceptible to red rot disease that results deterioration in both yield of cane and sugar recovery. This phenomenon is occurred due to the use of traditional planting materials, sets. Adaptation of tissue culture method brought about substantial improvement in propagules free from diseases and grew vigorously, which contributes to a greater productivity. Micropropagation ensures true to type and rapid multiplication of disease free material and as such can be used as super elite seed for quick spread of new variety.
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