Abstract: In vitro culture of lateral buds of field grown mature plants were tested in MS medium supplemented with different concentrations of BAP and NAA. Seasonal endophytic contamination was suppressed by shaking propagules for 2 h in 300 mg L-1 rifampicin before surface sterilization. Maximum survival rate was found in MS medium supplement with 1.0 mg L-1 BAP plus 0.20 mg L-1 NAA. The highest number of shoots was produced in MS medium containing 0.50 mg L-1 BAP and 0.20 mg L-1 NAA. Both the highest multiplication rate and the longest shoot were found in MS medium supplemented with 0.50 mg L-1 BAP plus 0.20 mg L-1 NAA. On the same treatment an increasing trend was observed in multiplication rate of shoot upto 5th subculture which decreased thereafter. First subculture produced the highest shoot length which decreased with the increase in number of subculture. Half strength MS medium supplemented with 1.0 mg L-1 IBA was found as the best treatment to produce root in the culture media. Rooted plantlets were transplanted successfully.
INTRODUCTION
Papaya (Carica papaya L.), an economically important crop of the tropics and sub tropics, is commercially propagated by seed. Since papaya is a polygamous species, many forms of inflorescences have been reported by many authors (Reuveni et al., 1991). Papaya is conventionally propagated by seed and therefore cultivation is hindered by problems due to the sex reversal, inherent heterozygosity and dioeciously nature of the crop (Veerannale, 1984; Rajeevan and Pandey, 1986). To avoid these problems tissue culture propagation could offer a valuable and a reliable procedure for propagation of papaya. Litz and Conover (1978 and 1981), Drew (1988), Winner (1988) and Rahman et al. (1992) reported a procedure based on shoot tips of field grown mature trees. The unbranched nature of papaya limits supply of shoot tips for initial culture. Moreover, excision of shoot tips from adult plant is hazardous and often results in death. This problem can be overcome by using lateral bud as explants for in vitro propagation. In our country, protocol has been established from papaya seedling but there is no recognized protocol for in vitro regeneration of papaya from mature plants and in this study, attempts have been made for rapid tissue culture of papaya by using lateral buds from mature field grown plants to establish suitable protocol for rapid plant regeneration in vitro from mature plants.
MATERIALS AND METHODS
The experiment was conducted in the Tissue Culture Laboratory of Biotechnology Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur during June 2004 to March 2005. Lateral buds consisting of the top young unexpended leaves collected from field grown mature plants were used as explants in this study. The buds were shaken in an antibiotic, rifampicin (RIF) at 300 mg L-1 to suppress the bacterial contamination. The buds were then cleaned with soap water and treated for 7 min with mercuric chloride (0.1%) containing a few drops of Tween-20. This was followed by 3-4 washings with sterile distilled water. The explants were agitated in sterile distilled water for 2-3 h in horizontal shaker at 160 rpm before inoculation to minimize the flow of latex into medium. The MS (Murashige and Skoog, 1962) was used as basal medium with different concentration of BAP (0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0 and 4.0 mg L-1) with different concentration of NAA (0.10, 0.20 and 0.30 mg L-1). Individual elongated shoots were excised carefully and transferred to half strength MS media with Indole-3-Butyric Acid (IBA) at different concentrations (0.10-0.5 mg L-1). The pH of the media was adjusted at 5.8 prior to autoclave and 8 g L-1 agar was added to solidify the media. The cultures were kept under approximately 2000 lux fluorescent light at 25±1°C with 16 h photoperiod.
RESULTS AND DISCUSSION
Establishment: A high rate of contamination was appeared as the cloud like growth at the base of the explants at the establishment stage of the explants. Shaking the lateral buds with RIF (300 mg L-1) was found to be most efficient in reducing contamination. Lateral buds shaken in rifampicin increased the uncontaminated cultures to 50% (Fig. 1). Reuveni et al. (1990) first applied FIR in suppressing bacterial contamination I culture of papaya and contamination rate reduced to 20%. RIF has also been reported in suppressing bacterial contamination in culture of other plant species (Bastiaens et al., 1983; Phillips et al., 1981; Young at al., 1984). Buds survived and grew better at 0.50 and 1.0 mg L-1 BAP with all level of NAA (Table 1). Maximum number of surviving buds (53.33%) was recorded at 1.0 mg L-1 BAP supplemented with 0.20 mg L-1 NAA. Maximum number of explants produced shoot (31.67%) in MS medium supplemented with 0.50 mg L-1 BAP plus 0.20 mg L-1 NAA. New growth was visible after 3-4 weeks of culture bud. The buds were considered to be established only when new growth spread approximately 1-2 cm diameter within 30-40 days. Bud cultured appeared very compact with highly shortened internodes and reduced leaf lamina (Fig. 2). The buds were transferred with a basal cut to fresh establishment. The buds were transferred with a basal cut to fresh establishment medium for accelerating growth.
Multiplication of shoots from buds established in culture: Individual
shoots were excised from the proliferating culture and subcultured into fresh
MS medium supplemented with different concentration of BAP (0.50-2.0 mg L-1)
along with different concentration of NAA (0.10-0.3 mg L-1). The
first subculture was made after 90 days of cultures and the subsequent culture
at an interval of 20 days. Total six subcultures were tested and the treatment
MS medium supplemented with 0.50 and 0.20 mg L-1 was found to be
the best for shoot multiplication and shoot length (Table 2
and 3).
| Fig. 1: | Percentage of uncontaminated cultures of papaya explants following 2 h soaking treatment with rifampicin (RIF 300 mg L-1) |
| Table 1: | Effect of growth regulators on the establishment of lateral bud explants |
| *Data after 30 days of inoculation. Within a column means followed by same letter(s) did not differ significantly at 1% level | |
An increasing trend in shoot multiplication rate was observed with the increase
in subculture number upto 5th subculture which decreased there after. In the
present study, average 12.73 fold shoot multiplication rate and 2.83 cm shoot
length was observed (Table 4). The highest shoot length was
found from 1st subculture (Fig. 3) which slightly decreased
in the subsequent cultures. With addition of BAP and NAA in the MS medium Litz
and Conover (1978), Hossain et al. (1993), Winner (1988) and Bhuyain
and Akond (2000) obtained almost similar result.
| Table 2: | Effect of growth regulators on multiplication rate at different in papaya |
| Within a column means followed by same letter(s) did not differ significantly at 1% level | |
| Table 3: | Effect of growth regulators on shoot length at different subculture in papaya |
| Within a column means followed by same letter(s) did not differ significantly at 1% level | |
| Fig. 2: | Growth of lateral bud in establishment medium (0.50 mg L-1 BAP + 0.20 mg L-1 NAA) after 40 days of incubation |
Rooting: In this study for rooting different concentrations IBA (0.10,
0.20, 0.30, 0.40, 0.05, 1.0, 1.5 and 2.0 mg L-1) in half strength
MS medium were tested for rooting but 0.10, 0.20, 0.30, 0.40 and 2.0 mg L-1
IBA produced no root.
| Fig. 3: | Multiple shoot in 1st subculture in MS medium containing 0.50 mg L-1 BAP + 0.20 mg L-1 NAA |
Half strength MS supplemented with 1.0 mg L-1 IBA gave the best
result for all the characters studied (Table 5). The treatment
1.5 mg L-1 IBA produced very few numbers of roots with very short
length.
| Fig. 4: | Rooted plantlet in half MS supplemented with 1.0 mg L-1 IBA |
| Fig. 5: | A papaya seedling produced from in vitro culture of lateral bud |
Large callus at the base of the shoot was found and no plantlet survived from
this concentration. Root of elongated shoots was obtained at relatively high
frequency when IBA at 1.0 mg L-1 was incorporated in the medium (Fig.
4). Litz and Conover (1978) recommended rooting to be carried out on half
strength MS supplemented with NAA (0.10-1.5 mg L-1) or IBA (0.10-1.0
mg L-1), but IBA induced the best result.
| Table 4: | Multiplication rate of shoots at different subcultures in MS medium supplemented with 0.50 mL-1 BAP and 0.20 mL-1 |
| *Mean±standard deviation | |
| Table 5: | Effect of IBA on rooting of papaya shoots |
| Within a column means followed by same letter(s) did not differ significantly at 1% level | |
Rooted plantlets were transplanted to plastic pot containing mixture of compost and soil and covered with polythene to protect from dehydration. After hardening they were transferred to net house (Fig. 5) until planted in the soil.
CONCLUSIONS
In our laboratory, this experiment became helpful to develop a protocol for in vitro regeneration of papaya plant from mature plant using lateral bud. Although the survival rate of the plantlets were low, its improvement is possible for commercial purposes and the present study indicated the possibility of producing papaya plants from mature plants through later bud culture. In the culture media, formation of calli is a great problem to develop root and further study will help to remove the problem.