J. bio-sci.
16: 59-65, 2008 ISSN 1023-8654
http://www.banglajol.info/index.php/JBS/index
MICROPROPAGATION OF CUCURBITA MAXIMA DUCH. THROUGH SHOOT TIP CULTURE
F Mahzabin, S Parvez and M F Alam*
Biotechnology and Microbiology Laboratory
Department of Botany, University of Rajshahi, Rajshahi- 6205, Bangladesh
Abstract
Micropropagation of pumpkin (Cucurbita maxima Duch.) was achieved using shoot tip of in vitro grown
seed derived plants of two cultivars namely, Bikrompuri and Baromasi of Bangladesh. The excised shoot
tips were cultured on MS medium containing KIN, BA, NAA at various levels of concentration and
combination for shoot induction and proliferation, and best response was found at 3.0 mg/l of BA.
Shoots were rooted most effectively in ½ MS medium supplemented with 1.0 mg/l IBA. Bikrompuri was
found more responsive than Baromasi for rapid clonal propagation.
Key words: Micropropagation, Direct organogenesis, Shoot tip, Pumpkin
Introduction
Cucurbita genus consists of 13 species, including C. maxima Duch. (Pumpkin) is a widely cultivated
vegetable in East Asian countries, such as China, Japan and Korea and good sources of carbohydrate, fat,
protein, mineral salts and vitamins (Gopalan et al. 1982). Many human nutritional deficiencies and diseases
commonly encountered in developing countries are preventable by an intelligent and fulfil utilization of
vegetables. Therefore, genetic improvements are recommended for vegetable crops including pumpkin.
Although conventional breeding methods are most widely used for crop improvement, but there have some
potential limitations (Chaudhury 1994). Some of genetic potential is not available due to sexual
incompatibility barriers appearing in intergeneric and interspecific levels as observed in several crops
(Deakin et al. 1971, Niemirowicz-Szczytt and Kubicki 1979, Olmstead 1989). Pumpkin is a cross-pollinated
plant. So, it can not produce sufficient hybrid seeds because large scale emasculation is not possible. This
plant shows abrupt inbreeding depression after two generation of selfing.
The techniques of plant tissue culture and genetic manipulation are useful to solve those problems (Razdan
1993). Recent developments in biotechnology have opened up several ways for the crop breeding using
transformation in which heterologous genes can be introduced into exiting cultivars. In many instances,
however, the lack of an efficient regeneration system causes limitations on the use of gene transfer
technology for the vegetable crops. An efficient plant regeneration system is, therefore, essential for
transformation and propagation as well as for in vitro breeding through selection of somaclonal variation and
somatic hybridization. In comparison with cucumber (Cucumis sativus) (Monohiuddin et al. 1997) and
watermelon (Citrullus vulgaris) (Dong and Jia 1991, Dabauza et al. 1997), less attention still has been given
to tissue culture of Cucurbita species. Therefore, attempts were taken to develop efficient protocol for plant
regeneration through direct organogenesis of the studied genus.
Materials and Methods
The materials for the present investigation were comprised of two cultivars of pumpkin such as Bikrompuri
and Baromasi. Seeds of these cultivars were collected from “Sufola Biz Vandar”, BRAC centre, Gazipur and
* Corresponding author.
60 Mahzabin et al.
“Shyamoli Biz Vander” Udoyon Road, Chapai Nawabganj. Shoot tips (2-3 mm) isolated from the seed
derived in vitro grown plants of these cultivars were used for experimentation. MS medium with different
concentrations and combinations of KIN, BA, NAA with 3% sugar was used for shoot multiplication while ½
MS medium with different concentrations of IBA, NAA and IAA were used for root formation. The medium
was solidified with 6.2% agar.
Shoot tip were cut and carefully inoculated on the agar gelled semi-solid MS medium (Plate 1 a). All the
inoculated culture tubes were incubated in the growth chamber providing proper culture environment. After
multiple shoot formation, the plantlets were transferred to rooting media for sufficient root. The culture tubes
were checked regularly to observe the morphogenic response. When the regenerated plantlets produced
sufficient roots and attained a height of 7-9 cm were transferred in the soil and acclimatized.
Results and Discussion
In the present investigation different concentrations and combinations of cytokinins KIN and BA or a
combination of BA and NAA were used to see the response towards multiple shoot formation from shoot tip
of two pumpkin cultivars viz. Bikrompuri and Baromasi (Table 1).
In the present study among all the combinations and concentrations, the longest shoots (6.1±0.85 cm) and
the highest percentage of shoot formation (90.45%) were observed in treatment with 3.0 mg/l BA after 30
days of culture (Plate 1 b, c, d). However, the highest number of shoots per shoot tip (16.5±0.95) was noted
in the media containing 2.0 mg/l BA. Further increasing the concentration of BA did not improve the shoot
size and number. Several workers reported similar results in cucumber (Kathal et al. 1988; Misra and
Bhatnagar 1995) and in Cucurbita interspecific hybrid cultivar “Shintoza” (Sarowar et al. 2003). Whereas, in
case of MS0, the longest shoot (2.5±0.55 cm), number of shoots per shoot tip (2.1±0.12) and highest
percentage of shoot formation (10%) were not performed well, thus suggested the necessity of using
phytohormone for effective shoot formation and multiplication. Increased concentration of BA reduced shoot
length and promoted massive base callus. Khan et al. (1994) obtained similar results at 2.0 mg/l BA for
Chrysanthemum morifolium.
When BA was tested in combination with NAA and KIN result shows that single use of BA is more effective
than combination treatment with NAA and KIN for direct organogenesis from shoot tips in Cucurbita maxima.
Although the combination of 3.0 mg/l BA +0.2 mg/l NAA shows high (80.10%) percentage of shoot formation,
but the shoot number (14.1±0.32) and length (5.8±0.75 cm) were not increased. Single use of KIN was also
not encouraging. This is further supported in Figs. 1, 2 and 3 when the data from Table 1 was calculated
combinedly. This finding is similar to Sarowar et al. (2003). The superiority of BA over other cytokinins, auxin
and gibberillin for shoot multiplication has been reported by other investigators (Vieitez and Vieitez 1980;
Misra and Chaturvedi 1984, Lim-Ho and Kong 1985, Mhatre et al. 1985, Arulpragasem and Latiff 1986,
Benjamin et al. 1987 and Amin et al. 1997).
The combined result presented in Fig. 4 calculated from Table 1 further shows that Bikrompuri was more
responsive for multiplication of plantlets than Baromasi; thus proven differential genotypic performance in in
vitro system (Zelcer et al. 1984, Capote et al. 2000 and EL-Bakry 2002).
Micropropagation of Cucurbita 61
Table 1. Shoot multiplication from isolated shoot tips in MS medium contained different concentrations and
combinations of phytohormone. In each treatment 10 explants were used. Data were recorded
after 21 days of inoculation.
Cultivars
Treatment
Bikrompuri Baromasi
Explants Mean No. Mean length Explants Mean No. Mean length
induced of shoots/ of longest induced of shoots/ of longest
(mg/l)
multiple Culture shoot (cm) multiple Culture shoot (cm)
shoots (%) ±SE ±SE shoots (%) ±SE ±SE
MS0 (control) 10.00 2.1± 0.12 2.5± 0.55 9.11 2.0± 0.55 2.1± 0.75
1.0 35.66 5.3 ±0.33 4.7 ±0.27 28.00 3.7 ± 0.05 4.2 ±0.59
1.5 40.95 6.1 ±0.30 5.0 ±0.82 40.45 5.2 ±0.94 4.8 ±0.33
KIN 2.0 50.37 6.1 ±0.36 4.9 ±0.17 48.42 5.9 ±0.32 4.7 ±0.53
3.0 48.75 5.9 ±0.35 5.1 ±0.16 42.97 5.5 ±0.23 4.8 ±0.24
4.0 42.50 5.6 ±0.24 4.9 ±0.31 39.55 5.1 ±0.85 4.1 ±0.61
1.0 66.12 8.9 ±0.64 3.1±0.11 55.45 7.1±0.29 2.8±0.29
2.0 75.25 16.5±0.95 5.2±0.11 67.54 13.1±0.64 4.9±0.12
BA 3.0 90.45 14.1±0.65 6.1±0.85 85.75 11.2±0.64 5.8±0.22
4.0 80.58 12.8±0.29 4.8±0.12 75.36 8.5±0.51 4.2±0.12
5.0 70.64 10.9±0.14 2.2±0.11 65.45 9.1±0.29 2.0±0.07
1.0+2.0 45.15 6.9±0.84 4.5±0.54 32.75 6.8±0.58 4.7±0.12
2.0+2.0 50.33 7.2±0.55 5.1±0.66 45.85 7.4±0.19 5.4±0.64
BA+KIN 3.0+2.0 60.66 6.8±0.33 5.5±0.66 58.10 7.1±0.28 5.6±0.21
4.0+2.0 48.72 6.1±0.78 5.1±0.32 35.00 6.9±0.14 5.3±0.11
5.0+2.0 40.25 5.8±0.50 4.5±0.38 30.55 5.9±0.91 4.8±0.25
1.0+0.2 60.15 8.12±0.35 5.1±0.39 55.12 8.8±0.52 4.9±0.55
2.0+0.2 70.45 11.0±0.21 5.4±0.15 65.25 10.1±0.11 5.1±0.63
BA+NAA 3.0+0.2 80.10 14.1±0.32 5.8±0.75 75.15 9.7±0.67 5.5±0.32
4.0+0.2 65.75 10.9±0.31 5.5±0.11 60.10 8.6±0.40 5.1±0.22
5.0+0.2 59.25 8.5±0.19 5.1±0.05 54.19 7.9±0.33 4.9±0.58
80 12
70
Number of shoots/culture
10
60
8
50
40 6
30
4
20
2
10
0 0
KIN BA B A +NA A B A +KIN KIN BA B A +NA A B A +KIN
Phytohorm ones Phytohorm ones
Fig 1. Comparison among different kinds and combinations Fig 2. Comparison among different kinds and combinations of
of phytohormone on multiple shoot initiation (%). phytohormone on number of shoots/culture.
62 Mahzabin et al.
6
60
5
50
Length of shoot (cm)
4
Bikrompuri
40
Baromasi
3
30
2 20
1
10
0 0
KIN BA B A +NA A B A +KIN Explant induced Number o f Length o f sho o t
Phytohorm ones multiple sho o ts sho o ts/culture (cm)
(%)
Fig 3. Comparison among different kinds and combinations of Fig 4. Comparison between two genotypes on studied three
phytohormone on length of shoot (cm) shooting parameters.
Elongated shoots (<4 cm) started root initiation within 7-8 days of inoculation when cultured onto ½ MS
medium added with different concentrations of NAA, IBA and IAA individually. And distinct variation was
observed among the treatments and between the cultivars (Table 2). All the treatments induced roots, but
maximum rooting (93.11 %) and vigorous growth of plantlets were observed on medium containing 1.0 mg/l
IBA within 12-14 days of transfer (Plate 1e). This was in accord with earlier reports on Cucumis melo (Haque
et al. 1984, Kathal et al. 1988, 1994, Singh et al. 1990 and Khalekuzzaman et al. 2003). However, the
highest mean number of roots (11.66±2.05) and highest mean length of longest root (7.75±0.93 cm) was
recorded in 2.0 mg/l of IBA. The figures 5, 6 and 7 prepared from combined data of Table 2 show that IBA is
more effective than NAA and IAA for root formation and proliferation. Similar to shoot, good response for root
formation and proliferation was observed in cultivar Bikrompuri than Baromasi (Fig. 8).
The successful establishment of plantlets in soil (Plate 1 f) suggested that shoot tips could be used for rapid
direct organogenesis in Cucurbita maxima.
Table 2. Proliferation of adventitious roots from in-vitro grown microclones of two pumpkin cultivars in ½ MS medium
with various concentrations of IBA, NAA and IAA singly. Data were recorded after 15 days of inoculation.
Cultivars
Treatment
Bikrompuri Baromasi
Mean no of Mean length Mean no of Mean length
Root Root
(mg/l) roots/culture of root in cm root per of root in cm
formation (%) formation (%)
±SE) ±SE) culture ±SE) ±SE)
½ MS0 5.0 2.50±0.95 0.85±1.15 5.1 1.9±0.85 0.80±1.11
0.5 58.33 11.12±1.05 7.50±0.64 50.17 10.11±1.04 7.23±0.64
1.0 93.11 8.66±1.74 6.08±0.33 87.45 7.90±1.12 5.91±0.30
IBA
2.0 76.25 11.66±2.05 7.75±0.93 67.54 10.33±1.95 7.10±0.85
3.0 66.35 7.12±1.55 7.25±0.74 59.33 6.75±1.23 7.14±0.71
0.5 50.22 6.95±0.61 5.01±0.25 45.11 5.10±0.57 4.80±0.23
1.0 70.32 8.11±2.11 6.00±0.78 60.14 7.01±1.25 5.10±0.43
NAA
2.0 50.15 7.5±1.15 5.03±0.54 40.22 6.23±01 4.50±0.32
3.0 40.45 5.12±0.25 4.80±0.63 30.44 4.30±0.23 3.90±0.12
0.5 60.13 4.50±0.51 8.50±0.64 57.33 4.10±0. 41 7.91±0.55
1.0 64.32 5.61±0.67 9.00±0.75 59.60 5.01±0.61 8.10±0.61
IAA
2.0 50.85 4.10±0.04 7.71±0.51 50.33 3.5±0.31 6.70±0.17
3.0 40.85 3.80±0.31 6.10±0.71 30.35 2.90±0.21 5.50±0.30
Micropropagation of Cucurbita 63
80 10
Mean no of roots/culture
70 9
Mean no of root/culture
8
Root formation (%)
60
7
50 6
40 5
30 4
3
20
2
10 1
0 0
IBA NAA IAA IBA NAA IAA
Phytohorm one s Phytohorm one s
Fig 5. Comparison among different kinds of auxin on multiple root Fig 6. Comparison among different kinds of auxin on number of
formation (%). roots/culture.
8 70
7
Mean length of root (cm)
60
6
50
5 Bikrompuri
40
4 Baromasi
30
3
2 20
1 10
0 0
IBA NAA IAA Root Mean no of Mean length
formation roots/culture of root (cm)
Phytohorm one s
(%)
Fig 7. Comparison among different kinds of auxin on length of Fig 8. Comparison between two genotypes on studied three rooting
root (cm) parameters.
Plate 1. Figures a-f showing different stages of culture for multiple shoot formation and acclimatization of plants derived from shoot tip
explant in Cucurbita maxima. a. Inoculation of excised shoot tip. b. Development of shoot tip after 4 days of inoculation. c. Proliferation
of multiple shoots after 15 days of inoculation, d. Farther proliferation of multiple shoot after 30 days of inoculation. e. Development of
rootlets in excised shoot. f. Acclimatization of plantlet in soil.
64 Mahzabin et al.
References
Amin M N, Azad M A K and Alam S M M (1997) Regeneration of plantlets in vitro from a medicinal shrub-Basak. 9th
Biennial Bot. Conf. (Dhaka Univ. Jan. 8-9) p. 31.
Arulpragasem and Latiff R (1986) Studies on the tissue culture of tea (Camellia sinensis (L.) O. Kuntze): A development
of a culture method for the multiplication of shoots. Srilanka J. Tea Sci. 55: 44-47.
Benjamin B D, Roja P C, Heble M R and Chanda M S (1987) Multiple shoot cultures of Atropa belledona: Effect of
physio-chemical factors on growth and alkaloid formation. J. Plant Physiol. 129: 129-135.
Capote R A, Fundora M Z and Perez D O (2000) Effect of different factors on the in vitro plant regeneration from leaflets
of five genotypes of tomato (Lycopersicon esculentum Mill.). Revista del Jardin Botanec Nacional 21: 71-76.
Chaudhury R C (1994) Introduction to plant breeding. Oxford and IBH Pub. Co. Pvt. Ltd. New Delhi, India.
Dabauza M, Bordas M, Salvador A and Roig L A (1997) Plant regeneration and Agrobacterium-mediated transformation
of cotyledon explant of Citrullus colocynthis (L.) Schrad. Plant Cell Rep. 16: 888-892.
Deakin J R, Bohn G W and Whitaker T W (1971) Interspecific hybridization in Cucumis. Econ. Bot. 25: 195-211.
Dong J Z and Jia S R (1991) High frequency plant regeneration from cotyledon explant of watermelon (Citrullus vulgaris
Schrad.). Plant Cell Rep. 9: 559-562.
EL-Bakry A A (2002) Effect of genotype, growth regulators, carbon source and pH on shoot induction and regeneration in
tomato. In vitro Cellular and Developmental Biology. Plant 38(5): 501-507.
Gopalan C, Rama S B V and Balasubramanian S C (1982) Nutritive value of Indian foods. Indian Council of Medical
Research, National Institute of Nutrition, Hyderabad.
Haque M I, Hoque M M, Begum A and Islam A S (1984) In vitro regeneration of plantlets from different explants of V.
mungo L. Hepper Bangladesh J. Bot. 13(1): 45-56.
Kathal R, Bhatnagar S P and Bhojwani S S (1988) Regeneration of plant of leaf explants of Cucumis melo L. cv. Pusa
Sharbati. Plant Cell Rep. 7: 449-451.
Kathal R, Bhatnagar S P and Bhojwani S S (1994) Plant regeneration from the callus derived from root explants of
Cucumis melo L. cv. Pusa Sharbati. Plant Sci. 96: 137-142.
Khalekuzzaman M, Alam M F, Nuruzzaman M, Begum N, Ahmed M G, Islam M A and Joarder O I (2003) Clonal
propagation of Averrhoa carambola Linn. through nodal culture of mature tree. J. bio-sci. 3(12): 1153-1157.
Khan M, Khanam A D, Ara A K and Hossain A K M (1994) In vitro plant regeneration in Chrysanthemum morifolium
Ramat. Plant Tissue Cult. 4(1): 53-57.
Lim-Ho C L and Kong L S (1985) Micropropagation of Lagerstroemia speciosa (L.) Pers. (Lythraceae). The Gardens
Bulletin Singapore 38(2): 175-184.
Mhatre M, Bapat N A and Rao P S (1985) Micropropagation and protoplast culture of peanut (Arachis hypogaea L.). Curr.
Sci. 54: 1052-1056.
Misra A K and Bhatnagar S P (1995) Direct shoot regeneration from the leaf explants of cucumbar (Cucumis sativas L.).
Phytomorphology 45: 47-55.
Misra P and Chaturvedi H C (1984) Micropropagation of Rosmarinus officinalis L. Plant Cell, Tissue Organ Cult. 3: 163-168.
Monohiuddin A K M, Chowdhury M K U, Abdullah Z C and Napis S (1997) Influence of silver nitrate (Ethylene inhibitor)
on cucumber in vitro shoot regeneration. Plant Cell, Tissue Organ Cult. 51: 75-78.
Micropropagation of Cucurbita 65
Niemirowicz-Szezytt K and Kubicki B (1979) Cross-fertilization between cultivated species of genera Cucumis L. and
Cucurbita L. Genet. Pol. 20: 117-125.
Olmstead R G (1989) The origin and function of self-incompatibility in flowering plants. Sex Plant Rep. 2: 127-136.
Razdan M K (1993) Application to horticulture and forestry. In: An introduction to plant tissue culture. Oxford and IBH
Publishing Co. Pvt. Ltd. New Delhi, India, pp. 245-263.
Sarowar S, Oh H Y, Hyung N I, Min B W, Harn CH, Yang S K, Ok S H and Shin J S (2003) In vitro micropropagation of a
Cucurbita interspecific hybrid cultivar – a root stock plant. Plant Cell, Tissue Organ Cult. 75: 179-182.
Singh M N, Kathal R and Bhatnagar S P (1990) Regeneration of plants from hypocotyl and cotyledon culture of Cucumis
melo L. cv. Pusa madhuras. Phytomorphology 40: 401-405.
Vieitez A M and Vieitez E (1980) Plantlet formation from embryonic tissue of chestnut grown in vitro. Plant Physiol.
50: 127-130.
Zelcer A, Soferman O and Izhar S (1984). An in vitro screening for tomato genotypes exhibiting efficient shoot
regeneration. J. Plant Physiol. 115(3): 211-215.
