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In vitro and in vivo germination of papaya (Carica papaya L.) seeds
Scientia Horticulturae 91 (2001) 39±49
In vitro and in vivo germination of papaya (Carica papaya L.) seeds J. Bhattacharya, S.S. Khuspe* Plant Tissue Culture Division, National Chemical Laboratory, Pune 411 008, India Accepted 19 December 2000
Abstract The effect of various pre-sowing treatments on seed germination in soil of 10 widely cultivated cultivars of papaya were studied. Intact seeds, naked embryos, halved seeds and halved embryos were germinated under in vitro culture conditions and the effects of thidiazuron (TDZ), 6-benzyl amino purine (BAP), naphthalene acetic acid (NAA), 2,4-dichloro phenoxyacetic acid (2,4-D) and 2,4,5-trichloro phenoxyacetic acid (2,4,5-T) at different concentrations, temperature and light were examined. Without pretreatment, the percentage germination in soil varied between 3 and 71% (average 40.2%). Soaking for 24 h in 200 ppm GA3 increased germination to 12±79% (average 56.5%). The difference between in vivo and in vitro seed germination was observed to be the lowest in honeydew (6.3%) and highest in Disco (68%). In general, in vitro culture conditions increased the percentage and rate of seed germination in all cultivars. An average maximum germination of 95.5% was obtained after 7±8 days for naked embryos when cultured in the light at 308C on Murashige and Skoog's medium (MS) supplemented with TDZ (1.0 mM/l). # 2001 Elsevier Science B.V. All rights reserved. Keywords: Papaya; Seed; Germination; Carica papaya L.; In vitro germination; In vivo germination
Abbreviations: BAP, 6-Benzyl amino purine; GA3, Gibberellic acid; NAA, Naphthalene acetic acid; MS, Murashige and Skoog medium; TDZ, Thidiazuron; 2,4-D, 2,4-Dichloro phenoxyacetic acid; 2,4,5-T, 2,4,5-Trichloro phenoxyacetic acid * Corresponding author. Tel.: 91-020-5893300/5893400, ext.: 2218; fax: 91-020-349038. E-mail address: [email protected] (S.S. Khuspe). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 2 3 7 - 0
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J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
1. Introduction Papaya (Carica papaya L.) is a tropical and subtropical crop and is considered to be one of the most important sources of vitamins A and C and the industrially important enzyme papain. The world-wide production of papaya in 1998 was 4,801,093 Mt with a total area under cultivation of 261 kha. Conventionally, papaya is propagated by seeds and has the disadvantage of heterogeneity as a result of cross-pollination. The productivity of the crop can be increased by growing elite cultivars produced by in vitro clonal propagation. Need for in vitro explants arose because the establishment of cultures from explants grown outdoors is dif®cult due to severe contamination problems. Although somatic embryogenesis has been reported in papaya (Fitch and Mansherdt, 1990; Monmarson et al., 1995; Cheng et al., 1987; Cabrera-Ponce et al., 1996), the whole process of developing a new plantlet takes almost 2±3 months. The production of the crop is also hampered by the papaya ring spot viral disease and breeders are under continuous pressure to produce disease resistant cultivars. Breeding programmes are further complicated by limited and variable seed germination. This problem becomes serious when seeds of hybrids are to be used where the seed yield is very low. To ensure seed survival and viability, a high germination percentage of these seeds is necessary. Germination of seeds in in vitro culture conditions has been reported in many crops such as cotton (Tort, 1996), bramble (Ke et al., 1985), Cypripedium (Depauw and Remphrey, 1993), Chaenorrhinum (Arnold et al., 1996), and neem (Murthy and Saxena, 1998). Improvement of seed germination through in vitro culture has been reported in banana (Pancholi et al., 1995). The effects of temperature, light, presoaking treatment, GA3, removal of seed coat and halving of seeds have earlier been reported to affect in vivo and/or in vitro germination of various crops (Shankarraja and Sulikeri, 1993; Page and Staden, 1985; Kyauk et al., 1995; Tseng, 1991; Ke et al., 1985). The present study was conducted to examine the importance of various factors that might affect the germination of C. papaya seeds under in vivo and/or in vitro culture conditions and to study the possible advantages of in vitro germination over direct sowing of seeds in soil. 2. Materials and methods 2.1. Material used Seeds of 10 cultivars of papaya, viz. Honey Dew, Pusa Majesty, Hybrid 781, Hybrid 786, Washington, Pusa Delicious, Disco, Co-4, Experiment 15 and Pant Papaya, were collected from the Regional Fruit Research station, Ganeshkhind, Pune, India.
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
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2.2. In vivo germination Prior to use, seeds were subjected to various pre-sowing treatments, i.e. soaking in water for 24 and 48 h, acid scari®cation with 0.1 N hydrochloric acid (HCl) (1 and 2 min), 0.1 N nitric acid (HNO3) (2 and 5 min), concentrated sulphuric acid (H2SO4) (30 and 60 s), soaking in gibberellic acid (GA3), NAA (100, 200 ppm each) and 0.1% potassium nitrate (KNO3). Treatment of GA3, NAA and KNO3was given for 24 h. All these experiments were carried out separately at room temperature. The seeds were not sterilised before use. The seeds were washed with tap water to remove traces of the chemicals and were sown directly in the soil at a depth of 3 cm in a tray. (Hereafter direct sowing of seeds in soil will be referred as in vivo condition.) Watering was done every 5 days. Germination of the seeds was tested at temperatures of 15, 20, 25, 30, 35 and 408C. Seeds were sown in three replications. Each replication contained 100 seeds. Radical emergence was the criterion for germination. The observations on germination were recorded at weekly intervals by taking the seeds out from the soil. Once germinated, the seedlings were removed and placed in the ®eld. 2.3. In vitro seed germination The seeds were kept under running water at room temperature for 1 h and then washed in liquid soap for 10 min. Seeds were then surface sterilised using ethyl alcohol (70%) for 30 s followed by treating with sodium hypochlorite (NaOCl) 4% for 10 s inside the laminar ¯ow cabinet. To remove traces of chlorine, the seeds were washed three times with sterile distilled water and soaked in water for about an hour. Four different types of explants were used, intact and halved seed and intact and halved embryos. Seeds were cut longitudinally and the white embryo was taken out aseptically and placed on MS medium (Murashige and Skoog, 1962) supplemented with a range of concentrations of BAP, NAA, 2,4-D, 2,4,5-T, TDZ (1±20 mM/l each). The basal medium consisted of full strength MS salts, 3% sucrose and 0.8% agar. The pH of the medium was adjusted to 5.8 and the medium was autoclaved for 20 min at 1218C. Thirty millilitres of the medium was poured in each 85-mm petridish. The experiment was laid out with three replications in a randomised block design. Each plate contained 10 explants and each replication contained 30 explants. All the dishes were sealed with ``Para®lm'' and kept at temperatures of 15, 20, 25, 30, 35 and 408C in both light (16 h photoperiod under warm white tubular ¯uorescent lamps at 25 mmol m 2 s 1) and dark conditions. Dark incubation was achieved by keeping the plates wrapped with silver foil in the dark incubator. The progress of germination was recorded for a period of 40 days at 7-day intervals under a safelight. Statistical analysis of the data was done using Fisher's analysis of
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J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
variance techniques for factorial experiments (Fisher, 1954). Treatment means were compared at 0.05 probability and critical difference (CD) was calculated (Chandel, 1993). 3. Results and discussion 3.1. In vivo seed germination When sown in soil, the commencement of seed germination (i.e. radical protrusion) was observed after 16±20 days, depending upon the cultivar. Emergence of radicals continued intermittently for up to 40 days. Over that time 3±71% germination (40.2%) was observed, depending upon the cultivar (Table 1). All the in vivo germinated seedlings developed into healthy plantlets. Among all the pre-sowing treatments given to enhance the papaya seed germination, soaking of seeds in 200 ppm GA3 for 24 h was found to be the best for all the cultivars except Experiment 15, where the best result was obtained with GA3 100 ppm (Table 2). Sen and Gunthi (1977) also reported that GA3 was the best pre-sowing treatment to be given to non-chilled seeds. Positive effect of GA3 for improving the papaya seed germination was also reported earlier (Nagao and Furutani, 1986; Tseng, 1991). GA3 is also found to affect positively the seed germination of other crops (Kuwahara et al., 1999; Zarad et al., 1997). The next best pre-sowing treatment in papaya was found to be acid scari®cation with 0.1 N HNO3 for 2 min. KNO3 and NAA were both found to be inhibitory to papaya seed germination. A similar result with KNO3 was observed by Sarita and Domingos (1999) with lime. Temperature strongly in¯uenced germination of papaya seeds. None of the cultivars germinated at a temperature of 158C (results not shown) and above 408C. Germination percentage was low at 208C and increased with increasing temperature to a maximum of about 80% at 308C. Above 308C, percentage germination decreased with temperature and was very low at 408C (Fig. 1). These ®ndings con®rm the ®ndings of Yahiro and Yoshitaka (1982). For most of the cultivars, 258C was found to be more suitable than 358C for seed germination. However this ®nding is in contrast with the ®ndings of Furutani and Nagao (1987) who reported that heating to 358C gave a higher percentage of seedling emergence of papaya seeds than heating to 258C in in vivo condition. 3.2. In vitro seed germination Germination of seeds kept in in vitro started within 5±9 days. The maximum in vitro germination percentage (77%) was observed with `Honey Dew', whereas `Pant Papaya' responded least (71.0%) in in vitro germination, after 40 days.
Table 1 Start of germination and percentage germination after 40 days of in vitro and in vivo papaya seed germinationa Days for commencement of germination In vivo
In vitro
Pusa Majesty Pinky Flesh Pusa Delicious Experiment 15 Disco Hybrid 781 Hybrid 786 Honey Dew Washington Pant Papaya
18:3 1:5 20:7 4:0 19:3 1:5 17:7 2:5 16:7 1:5 18:7 1:1 18:0 1:0 16:7 1:5 17:7 2:5 19:7 1:5
5:3 0:6 6:0 1:0 8:0 1:0 6:0 1:0 9:3 3:2 8:7 2:9 6:0 1:7 7:3 0:6 7:0 0:0 6:7 3:0
Mean
18.33
7.03
CD CD CD CD
for for for for
genotype conditions genotype condition treatment
Mean
17.2 15.8 18.5 18.0 22.5 21.7 20.2 18.8 18.8 22.0
4.5 35.5 2.0 5.8
Percentage germination after 40 days from sowing In vivo
In vitro
41:3 4:7 48:7 3:5 44:0 7:0 16:3 2:1 3:3 4:2 39:3 4:2 45:7 1:1 70:7 4:9 49:0 5:2 43:0 6:0
74:0 3:0 74:0 4:6 73:3 4:5 74:0 3:6 71:3 3:0 71:7 5:5 73:3 4:2 77:0 1:7 70:3 0:6 71:0 3:0
40.16
59.48
Mean
56.3 61.3 51.8 42.7 28.7 48.3 47.0 69.8 52.0 54.0
15.38 38.64 21.76 20.13
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
Cultivar used
a
CD: critical difference at 5% level of signi®cance, : standard error at 38 degrees of freedom. Explant used: intact seed. Conditions were 308C and 16 h photoperiod.
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Table 2 Percentages in vivo papaya seed germination after various pre-sowing treatmentsa Cultivar used
Pusa Majesty Pinky Flesh Pusa Delicious Experiment 15 Disco Hybrid 781 Hybrid 786 Honey Dew Washington Pant Papaya a
Control
41:3 4:7 48:7 3:5 44:0 7:0 16:3 2:1 03:3 4:2 39:3 4:2 45:7 1:1 70:7 4:9 49:0 5:2 43:0 6:0
Water soaking
HCl scarification
HNO3 scarification
H2SO4 scarification
24 h GA3 dipping
24 h
48 h
1 min
2 min
2 min
5 min
30 s
60 s
100 ppm
200 ppm
45:0 6:0 55:7 4:0 67:0 1:0 17:7 2:5 04:3 4:9 42:7 2:5 49:0 6:0 70:0 0:0 51:7 2:9 45:0 5:0
48:0 2:0 58:0 2:6 47:7 4:6 19:3 8:1 05:0 4:3 39:6 6:6 51:0 3:5 73:3 1:5 53:7 6:0 47:0 5:3
17:3 1:5 18:0 4:6 30:0 3:0 02:7 2:5 01:3 1:5 31:7 4:2 32:7 2:0 32:3 4:0 36:3 3:0 35:7 4:2
18:7 1:1 14:3 3:2 53:0 2:0 02:6 2:1 02:0 1:0 27:7 2:1 51:0 4:0 36:7 5:0 42:3 5:0 34:7 1:5
60:0 2:0 58:7 2:1 66:7 3:2 09:7 2:5 06:0 2:6 38:7 3:5 54:0 4:6 65:7 5:1 56:7 5:7 49:0 3:6
11:7 2:9 11:3 3:2 16:7 1:5 10:0 2:0 09:3 1:1 06:7 1:5 11:7 2:9 10:0 0:0 13:7 1:5 11:7 1:5
7:0 1:7 5:3 0:6 7:7 2:5 4:3 1:1 6:3 1:1 4:7 0:6 5:0 0:0 6:7 2:9 9:3 4:9 7:7 2:5
6:0 2:6 3:3 1:5 6:3 3:0 3:0 1:0 3:0 2:6 1:7 1:5 4:0 1:0 3:0 0:0 5:3 4:2 2:3 0:6
50:0 0:0 56:0 3:6 48:0 3:6 21:7 3:0 08:3 2:8 45:0 1:0 52:7 2:5 70:7 2:5 56:7 2:1 48:3 5:8
70:3 4:0 61:3 2:1 67:3 4:5 21:0 5:3 12:3 1:5 54:3 5:8 62:7 2:5 79:0 4:6 69:3 4:0 67:3 2:5
CD: critical difference at 5% level of signi®cance, : standard error at 198 degrees of freedom. Explant used: intact seed. Conditions kept: 308C and 16 h photoperiod. CD for genotype: 5.3; treatment: 3.7; time: 2.3; genotype treatment interaction, genotype time interaction: non-signi®cant; treatment time interaction: 5.3; genotype, treatment and time interaction: non-signi®cant.
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
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Fig. 1. In vivo papaya seed germination at different temperatures (8C). Values in the X-axis represent the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya).
Of the four types of seed material (intact and halved seed and intact and halved embryo), intact embryo yielded the highest germination percentages (Table 3). All these seedlings arose directly from the zygotic embryos. No somatic embryogenesis was observed. The lowest percentage of seedlings was produced by halved embryos. Not all the germinated seeds developed into whole plants. Some (7±8%) produced roots with stunted shoots when the highest concentration of TDZ (20 mM/l) was used. A similar trend was also observed by Sanago et al. (1995) in geranium, where an increase in concentration of TDZ increased the number of stunted seedlings. However, germination percentage was found to be improved by the presence of TDZ in the culture medium (Fig. 2a) followed by NAA (Fig. 2b) and BAP (Fig. 2c). The presence of TDZ in the medium was found to have a positive effect on germination of various crops (Murthy and Saxena, 1998). The highest germination percentage was observed at the lowest TDZ concentration (1.0 mM/l). Treatment with the higher concentrations of BAP and NAA and all concentrations of 2,4-D and 2,4,5-T favoured callus growth. In our study, seeds germinated faster in the 16 h photoperiod than in darkness. In cotton, Tort (1996) concluded that illumination signi®cantly increased the germination percentage. In vitro conditions were found to quicken the process of seed germination over in vivo conditions by about 1 week. Not only that in vitro seed germination was more uniform compared to in vivo germination process. Halved seeds and embryos germinated faster in the light than in darkness (results not shown), however, the percentage of total germination was similar after 10 days with dark incubation (Table 3). Although the effect of different temperatures on papaya seed has been studied in in vitro conditions (data not shown), an average maximum germination of 83.0% was observed with intact seeds at 308C in a 16 h photoperiod in all cultivars.
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Cultivar used
Pusa Majesty Pinky Flesh Pusa Delicious Experiment 15 Disco Hybrid 781 Hybrid 786 Honey Dew Washington Pant Papaya a
Percentage germination of intact seed
Percentage germination of halved seed
Percentage germination of naked embryo
Percentage germination of halved embryo
Light
Dark
Light
Dark
Light
Dark
Light
Dark
74:0 3:0 74:0 4:6 73:3 4:5 74:0 3:6 71:3 3:0 71:7 5:5 73:3 4:2 77:0 1:7 70:3 0:6 71:0 3:0
70:7 2:1 73:7 1:1 63:0 4:0 71:3 3:2 56:3 4:0 61:0 5:3 56:7 4:7 64:3 4:0 55:7 4:0 60:0 5:0
84:7 2:5 89:0 5:6 77:3 3:8 83:7 5:0 72:0 1:7 70:0 2:0 65:3 2:5 75:0 0:0 70:7 3:8 69:3 6:0
81:3 2:3 86:0 4:0 74:7 2:5 83:0 3:6 68:0 3:0 75:3 3:5 70:7 4:1 75:7 4:5 65:0 5:0 67:3 2:5
97.0 95.7 97.7 94.0 92.0 95.3 96.3 100.0 94.0 93.3
95:3 2:5 91:7 1:5 96:0 1:0 89:7 3:5 85:3 3:5 92:7 2:5 95:0 1:0 99:0 1:0 91:3 3:2 90:0 4:3
69:7 5:0 71:7 5:8 67:33 2:5 73:7 3:2 65:0 5:0 62:3 3:0 63:7 4:0 66:3 3:2 63:0 2:0 62:7 2:5
65:0 5:0 66:7 5:7 66:0 3:6 70:3 2:5 61:0 3:6 57:7 2:5 59:0 5:3 61:7 2:9 54:3 5:1 53:7 2:9
2.6 3.0 2.1 1.7 5.6 1.5 2.1 0.0 5.3 2.9
CD: critical difference at 5% level of signi®cance, : standard error at 158 degrees of freedom. Conditions kept: 308C and 16 h photoperiod. CD for genotype: 6.6; treatment: 4.2; treatment and condition interaction: 3.0; genotype treatment interaction: 13.3; genotype condition interaction: nonsigni®cant; treatment condition interaction: 6.0; genotype, treatment and condition interaction: non-signi®cant.
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
Table 3 Percentage in vitro germination of different papaya seed material in light and in darknessa
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
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Fig. 2. (a) In vitro papaya seed germination at different TDZ concentrations. Values in the X-axis represent the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya). The values indicated are obtained at 308C and 16 h photoperiod condition. (b) In vitro papaya seed germination at different NAA concentrations. Values in the X-axis represent the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya). The values indicated are obtained at 308C and 16 h photoperiod condition. (c) In vitro papaya seed germination at different BAP concentrations. Values in the X-axis represents the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya). The values indicated are obtained at 308C and 16 h photoperiod condition.
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J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
4. Conclusion After studying the effect of light, temperature, pre-sowing treatments, types of explants, and various hormonal treatments, on germination of 10 cultivars of papaya the following can be concluded. Light hastens the germination process. Germination of papaya seeds started at 208C, was maximal at 308C and then declined with increasing temperature. Soaking of seeds in GA3 (200 ppm) resulted in the maximum germination percentage in soil for all the cultivars except Experiment 15. The maximum percentage of seed germinated in media supplemented with TDZ (1.0 mM/l) as compared to BAP and NAA. Higher concentrations of BAP and all concentrations of 2,4-D and 2,4,5-T induced callusing of the explants. Considering the results obtained, it can be concluded that in vitro conditions favoured germination more than the in vivo environment. The present study not only enables a large number of aseptic seedlings to be produced in a short period of time but it also helps to speed up papaya breeding programmes. Acknowledgements Financial support in the form of a research grant by the Department of Biotechnology (DBT), Government of India, is gratefully acknowledged. We are thankful to Dr. K.V. Krishnamurthy for providing the central facility of the tissue culture laboratory. References Arnold, R.M., Slyker, J.A., Gupta, T.H., 1996. Germination of Chaenorrhinum minus seeds in response to gibberellin treatments. J. Plant Physiol. 148, 677±683. Cabrera-Ponce, J.L., Vegas-Garcia, A., Herrere-Estrella, L., 1996. Regeneration of transgenic papaya plants via somatic embryogenesis induced by Agrobacterium rhizogenes. In Vitro Cell Dev. Biol. P 32, 86±90. Chandel, S.R.S., 1993. A Handbook of Agricultural Statistics. Achal Prakashan Mandir, Kanpur, India, pp. B35±B36. Cheng, M.H., Wang, P.J., Maeda, E., 1987. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explant. Plant Cell Rep. 6, 348±351. Depauw, M.A., Remphrey, W.R., 1993. In vitro germination of three Cypripedium species in relation to time of seed collection, media and cold treatments. Can. J. Bot. 71, 879±885. Fisher, R.A., 1954. Statistical Methods for Research Workers, 12th Edition. Oliver & Boyd, Edinburgh, UK. Fitch, M.M., Mansherdt, R.M., 1990. Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.). Plant Cell Rep. 9, 320±324. Furutani, S.C., Nagao, M.A., 1987. In¯uence of temperature, KNO3 and GA3 and seed drying on emergence of papaya seedlings. Sci. Hort. 32, 67±72.
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
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Ke, S., Skirvin, R.M., Mcpheeters, K.D., Otterbachet, A.G., Galletta, G., 1985. In vitro germination and growth of Rubus seeds and embryos. Hort. Sci. 20, 1047±1049. Kuwahara, Y.C., Orika, O.E., MarchiLia, D.O.S., 1999. The effect of GA3 on Kiwi (Actinidia chinensis PI) seed germination. Sci. Agric. 56, 9±12. Kyauk, H., Hopper, N.W., Brigham, R.D., 1995. Effect of temperature and presoaking on germination, root length and shoot length of sesame (Sesamum indicum L.). Environ. Exp. Bot. 35, 345±351. Monmarson, S., Micchaux-Ferriere, N., Teisson, C., 1995. Production of high frequency embryogenic calli from integuments of immature seeds of Carica papaya L. J. Hort. Sci. 70, 57±64. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15, 473±497. Murthy, B.N.S., Saxena, P.K., 1998. Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.). Plant Cell Rep. 17, 469±475. Nagao, M.A., Furutani, S.C., 1986. Improving germination of papaya seed by density separation, potassium nitrate, and gibberellic acid. Hort. Sci. 21, 1439±1440. Page, Y.M., Staden, J.Y., 1985. In vitro propagation of Dierama latifolium. Hort. Sci. 20, 1049± 1050. Pancholi, N., Wetten, A., Caligari, P.D.S., 1995. Germination of Musa velutina seeds: comparison of in vivo and in vitro systems. In vitro Cell Dev. Biol. Plantarum 31, 127±130. Sanago, M.M., Murch, S.J., Slimmon, T.Y., Krishnaraj, S., Saxena, P.K., 1995. Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium hortorum Bailey). Plant Cell Rep. 15, 205±211. Sarita, L., Domingos, R.J., 1999. The effect of gibberellins, cytokinins and potassium nitrate on Rangpur lime (Citrus limonia Osbeck) seed germination. Sci. Agric. 56, 111±115. Sen, S.K., Gunthi, P., 1977. Effect of presowing seed treatments on the germination and seedling growth in papaya. Food Farming Agric. 9, 144±146. Shankarraja, N.S., Sulikeri, G.S., 1993. Presowing treatments of seeds to improve germination in cardamom (Elettaria cardamomum L. Maton var minor Watt). J. Plantation Crops 21, 116±117. Tort, N., 1996. Effect of light, different growth media, temperature and salt concentrations on germination of cotton seeds (Gossypium hirsutum L. cv. Nazilli 87). J. Agron. Crop Sci. 176, 217±221. Tseng, T.M., 1991. The effect of GA3 concentration and time of treatment on the promotion of papaya seed germination. Guoli Taiwan Daxue Nongxueyuan Yanjiu Baogao 31, 30±39. Yahiro, M., Yoshitaka, H., 1982. Growth inhibitors in papaya (Carica papaya L.) seeds. Nettai Nogyo. 26, 63±67. Zarad, S.S., Hossny, Y.A., El-Bagoury, H.M., 1997. Effect of various treatments on seed germination and water application on vegetative growth of desert date (Balanite egyptica L.) seedlings grown in different soils. Egypt J. Physiol. Sci. 21, 147±159.
In vitro and in vivo germination of papaya (Carica papaya L.) seeds J. Bhattacharya, S.S. Khuspe* Plant Tissue Culture Division, National Chemical Laboratory, Pune 411 008, India Accepted 19 December 2000
Abstract The effect of various pre-sowing treatments on seed germination in soil of 10 widely cultivated cultivars of papaya were studied. Intact seeds, naked embryos, halved seeds and halved embryos were germinated under in vitro culture conditions and the effects of thidiazuron (TDZ), 6-benzyl amino purine (BAP), naphthalene acetic acid (NAA), 2,4-dichloro phenoxyacetic acid (2,4-D) and 2,4,5-trichloro phenoxyacetic acid (2,4,5-T) at different concentrations, temperature and light were examined. Without pretreatment, the percentage germination in soil varied between 3 and 71% (average 40.2%). Soaking for 24 h in 200 ppm GA3 increased germination to 12±79% (average 56.5%). The difference between in vivo and in vitro seed germination was observed to be the lowest in honeydew (6.3%) and highest in Disco (68%). In general, in vitro culture conditions increased the percentage and rate of seed germination in all cultivars. An average maximum germination of 95.5% was obtained after 7±8 days for naked embryos when cultured in the light at 308C on Murashige and Skoog's medium (MS) supplemented with TDZ (1.0 mM/l). # 2001 Elsevier Science B.V. All rights reserved. Keywords: Papaya; Seed; Germination; Carica papaya L.; In vitro germination; In vivo germination
Abbreviations: BAP, 6-Benzyl amino purine; GA3, Gibberellic acid; NAA, Naphthalene acetic acid; MS, Murashige and Skoog medium; TDZ, Thidiazuron; 2,4-D, 2,4-Dichloro phenoxyacetic acid; 2,4,5-T, 2,4,5-Trichloro phenoxyacetic acid * Corresponding author. Tel.: 91-020-5893300/5893400, ext.: 2218; fax: 91-020-349038. E-mail address: [email protected] (S.S. Khuspe). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 2 3 7 - 0
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J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
1. Introduction Papaya (Carica papaya L.) is a tropical and subtropical crop and is considered to be one of the most important sources of vitamins A and C and the industrially important enzyme papain. The world-wide production of papaya in 1998 was 4,801,093 Mt with a total area under cultivation of 261 kha. Conventionally, papaya is propagated by seeds and has the disadvantage of heterogeneity as a result of cross-pollination. The productivity of the crop can be increased by growing elite cultivars produced by in vitro clonal propagation. Need for in vitro explants arose because the establishment of cultures from explants grown outdoors is dif®cult due to severe contamination problems. Although somatic embryogenesis has been reported in papaya (Fitch and Mansherdt, 1990; Monmarson et al., 1995; Cheng et al., 1987; Cabrera-Ponce et al., 1996), the whole process of developing a new plantlet takes almost 2±3 months. The production of the crop is also hampered by the papaya ring spot viral disease and breeders are under continuous pressure to produce disease resistant cultivars. Breeding programmes are further complicated by limited and variable seed germination. This problem becomes serious when seeds of hybrids are to be used where the seed yield is very low. To ensure seed survival and viability, a high germination percentage of these seeds is necessary. Germination of seeds in in vitro culture conditions has been reported in many crops such as cotton (Tort, 1996), bramble (Ke et al., 1985), Cypripedium (Depauw and Remphrey, 1993), Chaenorrhinum (Arnold et al., 1996), and neem (Murthy and Saxena, 1998). Improvement of seed germination through in vitro culture has been reported in banana (Pancholi et al., 1995). The effects of temperature, light, presoaking treatment, GA3, removal of seed coat and halving of seeds have earlier been reported to affect in vivo and/or in vitro germination of various crops (Shankarraja and Sulikeri, 1993; Page and Staden, 1985; Kyauk et al., 1995; Tseng, 1991; Ke et al., 1985). The present study was conducted to examine the importance of various factors that might affect the germination of C. papaya seeds under in vivo and/or in vitro culture conditions and to study the possible advantages of in vitro germination over direct sowing of seeds in soil. 2. Materials and methods 2.1. Material used Seeds of 10 cultivars of papaya, viz. Honey Dew, Pusa Majesty, Hybrid 781, Hybrid 786, Washington, Pusa Delicious, Disco, Co-4, Experiment 15 and Pant Papaya, were collected from the Regional Fruit Research station, Ganeshkhind, Pune, India.
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2.2. In vivo germination Prior to use, seeds were subjected to various pre-sowing treatments, i.e. soaking in water for 24 and 48 h, acid scari®cation with 0.1 N hydrochloric acid (HCl) (1 and 2 min), 0.1 N nitric acid (HNO3) (2 and 5 min), concentrated sulphuric acid (H2SO4) (30 and 60 s), soaking in gibberellic acid (GA3), NAA (100, 200 ppm each) and 0.1% potassium nitrate (KNO3). Treatment of GA3, NAA and KNO3was given for 24 h. All these experiments were carried out separately at room temperature. The seeds were not sterilised before use. The seeds were washed with tap water to remove traces of the chemicals and were sown directly in the soil at a depth of 3 cm in a tray. (Hereafter direct sowing of seeds in soil will be referred as in vivo condition.) Watering was done every 5 days. Germination of the seeds was tested at temperatures of 15, 20, 25, 30, 35 and 408C. Seeds were sown in three replications. Each replication contained 100 seeds. Radical emergence was the criterion for germination. The observations on germination were recorded at weekly intervals by taking the seeds out from the soil. Once germinated, the seedlings were removed and placed in the ®eld. 2.3. In vitro seed germination The seeds were kept under running water at room temperature for 1 h and then washed in liquid soap for 10 min. Seeds were then surface sterilised using ethyl alcohol (70%) for 30 s followed by treating with sodium hypochlorite (NaOCl) 4% for 10 s inside the laminar ¯ow cabinet. To remove traces of chlorine, the seeds were washed three times with sterile distilled water and soaked in water for about an hour. Four different types of explants were used, intact and halved seed and intact and halved embryos. Seeds were cut longitudinally and the white embryo was taken out aseptically and placed on MS medium (Murashige and Skoog, 1962) supplemented with a range of concentrations of BAP, NAA, 2,4-D, 2,4,5-T, TDZ (1±20 mM/l each). The basal medium consisted of full strength MS salts, 3% sucrose and 0.8% agar. The pH of the medium was adjusted to 5.8 and the medium was autoclaved for 20 min at 1218C. Thirty millilitres of the medium was poured in each 85-mm petridish. The experiment was laid out with three replications in a randomised block design. Each plate contained 10 explants and each replication contained 30 explants. All the dishes were sealed with ``Para®lm'' and kept at temperatures of 15, 20, 25, 30, 35 and 408C in both light (16 h photoperiod under warm white tubular ¯uorescent lamps at 25 mmol m 2 s 1) and dark conditions. Dark incubation was achieved by keeping the plates wrapped with silver foil in the dark incubator. The progress of germination was recorded for a period of 40 days at 7-day intervals under a safelight. Statistical analysis of the data was done using Fisher's analysis of
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J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
variance techniques for factorial experiments (Fisher, 1954). Treatment means were compared at 0.05 probability and critical difference (CD) was calculated (Chandel, 1993). 3. Results and discussion 3.1. In vivo seed germination When sown in soil, the commencement of seed germination (i.e. radical protrusion) was observed after 16±20 days, depending upon the cultivar. Emergence of radicals continued intermittently for up to 40 days. Over that time 3±71% germination (40.2%) was observed, depending upon the cultivar (Table 1). All the in vivo germinated seedlings developed into healthy plantlets. Among all the pre-sowing treatments given to enhance the papaya seed germination, soaking of seeds in 200 ppm GA3 for 24 h was found to be the best for all the cultivars except Experiment 15, where the best result was obtained with GA3 100 ppm (Table 2). Sen and Gunthi (1977) also reported that GA3 was the best pre-sowing treatment to be given to non-chilled seeds. Positive effect of GA3 for improving the papaya seed germination was also reported earlier (Nagao and Furutani, 1986; Tseng, 1991). GA3 is also found to affect positively the seed germination of other crops (Kuwahara et al., 1999; Zarad et al., 1997). The next best pre-sowing treatment in papaya was found to be acid scari®cation with 0.1 N HNO3 for 2 min. KNO3 and NAA were both found to be inhibitory to papaya seed germination. A similar result with KNO3 was observed by Sarita and Domingos (1999) with lime. Temperature strongly in¯uenced germination of papaya seeds. None of the cultivars germinated at a temperature of 158C (results not shown) and above 408C. Germination percentage was low at 208C and increased with increasing temperature to a maximum of about 80% at 308C. Above 308C, percentage germination decreased with temperature and was very low at 408C (Fig. 1). These ®ndings con®rm the ®ndings of Yahiro and Yoshitaka (1982). For most of the cultivars, 258C was found to be more suitable than 358C for seed germination. However this ®nding is in contrast with the ®ndings of Furutani and Nagao (1987) who reported that heating to 358C gave a higher percentage of seedling emergence of papaya seeds than heating to 258C in in vivo condition. 3.2. In vitro seed germination Germination of seeds kept in in vitro started within 5±9 days. The maximum in vitro germination percentage (77%) was observed with `Honey Dew', whereas `Pant Papaya' responded least (71.0%) in in vitro germination, after 40 days.
Table 1 Start of germination and percentage germination after 40 days of in vitro and in vivo papaya seed germinationa Days for commencement of germination In vivo
In vitro
Pusa Majesty Pinky Flesh Pusa Delicious Experiment 15 Disco Hybrid 781 Hybrid 786 Honey Dew Washington Pant Papaya
18:3 1:5 20:7 4:0 19:3 1:5 17:7 2:5 16:7 1:5 18:7 1:1 18:0 1:0 16:7 1:5 17:7 2:5 19:7 1:5
5:3 0:6 6:0 1:0 8:0 1:0 6:0 1:0 9:3 3:2 8:7 2:9 6:0 1:7 7:3 0:6 7:0 0:0 6:7 3:0
Mean
18.33
7.03
CD CD CD CD
for for for for
genotype conditions genotype condition treatment
Mean
17.2 15.8 18.5 18.0 22.5 21.7 20.2 18.8 18.8 22.0
4.5 35.5 2.0 5.8
Percentage germination after 40 days from sowing In vivo
In vitro
41:3 4:7 48:7 3:5 44:0 7:0 16:3 2:1 3:3 4:2 39:3 4:2 45:7 1:1 70:7 4:9 49:0 5:2 43:0 6:0
74:0 3:0 74:0 4:6 73:3 4:5 74:0 3:6 71:3 3:0 71:7 5:5 73:3 4:2 77:0 1:7 70:3 0:6 71:0 3:0
40.16
59.48
Mean
56.3 61.3 51.8 42.7 28.7 48.3 47.0 69.8 52.0 54.0
15.38 38.64 21.76 20.13
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
Cultivar used
a
CD: critical difference at 5% level of signi®cance, : standard error at 38 degrees of freedom. Explant used: intact seed. Conditions were 308C and 16 h photoperiod.
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Table 2 Percentages in vivo papaya seed germination after various pre-sowing treatmentsa Cultivar used
Pusa Majesty Pinky Flesh Pusa Delicious Experiment 15 Disco Hybrid 781 Hybrid 786 Honey Dew Washington Pant Papaya a
Control
41:3 4:7 48:7 3:5 44:0 7:0 16:3 2:1 03:3 4:2 39:3 4:2 45:7 1:1 70:7 4:9 49:0 5:2 43:0 6:0
Water soaking
HCl scarification
HNO3 scarification
H2SO4 scarification
24 h GA3 dipping
24 h
48 h
1 min
2 min
2 min
5 min
30 s
60 s
100 ppm
200 ppm
45:0 6:0 55:7 4:0 67:0 1:0 17:7 2:5 04:3 4:9 42:7 2:5 49:0 6:0 70:0 0:0 51:7 2:9 45:0 5:0
48:0 2:0 58:0 2:6 47:7 4:6 19:3 8:1 05:0 4:3 39:6 6:6 51:0 3:5 73:3 1:5 53:7 6:0 47:0 5:3
17:3 1:5 18:0 4:6 30:0 3:0 02:7 2:5 01:3 1:5 31:7 4:2 32:7 2:0 32:3 4:0 36:3 3:0 35:7 4:2
18:7 1:1 14:3 3:2 53:0 2:0 02:6 2:1 02:0 1:0 27:7 2:1 51:0 4:0 36:7 5:0 42:3 5:0 34:7 1:5
60:0 2:0 58:7 2:1 66:7 3:2 09:7 2:5 06:0 2:6 38:7 3:5 54:0 4:6 65:7 5:1 56:7 5:7 49:0 3:6
11:7 2:9 11:3 3:2 16:7 1:5 10:0 2:0 09:3 1:1 06:7 1:5 11:7 2:9 10:0 0:0 13:7 1:5 11:7 1:5
7:0 1:7 5:3 0:6 7:7 2:5 4:3 1:1 6:3 1:1 4:7 0:6 5:0 0:0 6:7 2:9 9:3 4:9 7:7 2:5
6:0 2:6 3:3 1:5 6:3 3:0 3:0 1:0 3:0 2:6 1:7 1:5 4:0 1:0 3:0 0:0 5:3 4:2 2:3 0:6
50:0 0:0 56:0 3:6 48:0 3:6 21:7 3:0 08:3 2:8 45:0 1:0 52:7 2:5 70:7 2:5 56:7 2:1 48:3 5:8
70:3 4:0 61:3 2:1 67:3 4:5 21:0 5:3 12:3 1:5 54:3 5:8 62:7 2:5 79:0 4:6 69:3 4:0 67:3 2:5
CD: critical difference at 5% level of signi®cance, : standard error at 198 degrees of freedom. Explant used: intact seed. Conditions kept: 308C and 16 h photoperiod. CD for genotype: 5.3; treatment: 3.7; time: 2.3; genotype treatment interaction, genotype time interaction: non-signi®cant; treatment time interaction: 5.3; genotype, treatment and time interaction: non-signi®cant.
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
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Fig. 1. In vivo papaya seed germination at different temperatures (8C). Values in the X-axis represent the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya).
Of the four types of seed material (intact and halved seed and intact and halved embryo), intact embryo yielded the highest germination percentages (Table 3). All these seedlings arose directly from the zygotic embryos. No somatic embryogenesis was observed. The lowest percentage of seedlings was produced by halved embryos. Not all the germinated seeds developed into whole plants. Some (7±8%) produced roots with stunted shoots when the highest concentration of TDZ (20 mM/l) was used. A similar trend was also observed by Sanago et al. (1995) in geranium, where an increase in concentration of TDZ increased the number of stunted seedlings. However, germination percentage was found to be improved by the presence of TDZ in the culture medium (Fig. 2a) followed by NAA (Fig. 2b) and BAP (Fig. 2c). The presence of TDZ in the medium was found to have a positive effect on germination of various crops (Murthy and Saxena, 1998). The highest germination percentage was observed at the lowest TDZ concentration (1.0 mM/l). Treatment with the higher concentrations of BAP and NAA and all concentrations of 2,4-D and 2,4,5-T favoured callus growth. In our study, seeds germinated faster in the 16 h photoperiod than in darkness. In cotton, Tort (1996) concluded that illumination signi®cantly increased the germination percentage. In vitro conditions were found to quicken the process of seed germination over in vivo conditions by about 1 week. Not only that in vitro seed germination was more uniform compared to in vivo germination process. Halved seeds and embryos germinated faster in the light than in darkness (results not shown), however, the percentage of total germination was similar after 10 days with dark incubation (Table 3). Although the effect of different temperatures on papaya seed has been studied in in vitro conditions (data not shown), an average maximum germination of 83.0% was observed with intact seeds at 308C in a 16 h photoperiod in all cultivars.
46
Cultivar used
Pusa Majesty Pinky Flesh Pusa Delicious Experiment 15 Disco Hybrid 781 Hybrid 786 Honey Dew Washington Pant Papaya a
Percentage germination of intact seed
Percentage germination of halved seed
Percentage germination of naked embryo
Percentage germination of halved embryo
Light
Dark
Light
Dark
Light
Dark
Light
Dark
74:0 3:0 74:0 4:6 73:3 4:5 74:0 3:6 71:3 3:0 71:7 5:5 73:3 4:2 77:0 1:7 70:3 0:6 71:0 3:0
70:7 2:1 73:7 1:1 63:0 4:0 71:3 3:2 56:3 4:0 61:0 5:3 56:7 4:7 64:3 4:0 55:7 4:0 60:0 5:0
84:7 2:5 89:0 5:6 77:3 3:8 83:7 5:0 72:0 1:7 70:0 2:0 65:3 2:5 75:0 0:0 70:7 3:8 69:3 6:0
81:3 2:3 86:0 4:0 74:7 2:5 83:0 3:6 68:0 3:0 75:3 3:5 70:7 4:1 75:7 4:5 65:0 5:0 67:3 2:5
97.0 95.7 97.7 94.0 92.0 95.3 96.3 100.0 94.0 93.3
95:3 2:5 91:7 1:5 96:0 1:0 89:7 3:5 85:3 3:5 92:7 2:5 95:0 1:0 99:0 1:0 91:3 3:2 90:0 4:3
69:7 5:0 71:7 5:8 67:33 2:5 73:7 3:2 65:0 5:0 62:3 3:0 63:7 4:0 66:3 3:2 63:0 2:0 62:7 2:5
65:0 5:0 66:7 5:7 66:0 3:6 70:3 2:5 61:0 3:6 57:7 2:5 59:0 5:3 61:7 2:9 54:3 5:1 53:7 2:9
2.6 3.0 2.1 1.7 5.6 1.5 2.1 0.0 5.3 2.9
CD: critical difference at 5% level of signi®cance, : standard error at 158 degrees of freedom. Conditions kept: 308C and 16 h photoperiod. CD for genotype: 6.6; treatment: 4.2; treatment and condition interaction: 3.0; genotype treatment interaction: 13.3; genotype condition interaction: nonsigni®cant; treatment condition interaction: 6.0; genotype, treatment and condition interaction: non-signi®cant.
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
Table 3 Percentage in vitro germination of different papaya seed material in light and in darknessa
J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
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Fig. 2. (a) In vitro papaya seed germination at different TDZ concentrations. Values in the X-axis represent the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya). The values indicated are obtained at 308C and 16 h photoperiod condition. (b) In vitro papaya seed germination at different NAA concentrations. Values in the X-axis represent the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya). The values indicated are obtained at 308C and 16 h photoperiod condition. (c) In vitro papaya seed germination at different BAP concentrations. Values in the X-axis represents the average germination percentage of all cultivars (Pusa Majesty, Pinky Flesh, Pusa Delicious, Experiment 15, Disco, Hybrid 781, Hybrid 786, Honey Dew, Washington, Pant Papaya). The values indicated are obtained at 308C and 16 h photoperiod condition.
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J. Bhattacharya, S.S. Khuspe / Scientia Horticulturae 91 (2001) 39±49
4. Conclusion After studying the effect of light, temperature, pre-sowing treatments, types of explants, and various hormonal treatments, on germination of 10 cultivars of papaya the following can be concluded. Light hastens the germination process. Germination of papaya seeds started at 208C, was maximal at 308C and then declined with increasing temperature. Soaking of seeds in GA3 (200 ppm) resulted in the maximum germination percentage in soil for all the cultivars except Experiment 15. The maximum percentage of seed germinated in media supplemented with TDZ (1.0 mM/l) as compared to BAP and NAA. Higher concentrations of BAP and all concentrations of 2,4-D and 2,4,5-T induced callusing of the explants. Considering the results obtained, it can be concluded that in vitro conditions favoured germination more than the in vivo environment. The present study not only enables a large number of aseptic seedlings to be produced in a short period of time but it also helps to speed up papaya breeding programmes. Acknowledgements Financial support in the form of a research grant by the Department of Biotechnology (DBT), Government of India, is gratefully acknowledged. We are thankful to Dr. K.V. Krishnamurthy for providing the central facility of the tissue culture laboratory. References Arnold, R.M., Slyker, J.A., Gupta, T.H., 1996. Germination of Chaenorrhinum minus seeds in response to gibberellin treatments. J. Plant Physiol. 148, 677±683. Cabrera-Ponce, J.L., Vegas-Garcia, A., Herrere-Estrella, L., 1996. Regeneration of transgenic papaya plants via somatic embryogenesis induced by Agrobacterium rhizogenes. In Vitro Cell Dev. Biol. P 32, 86±90. Chandel, S.R.S., 1993. A Handbook of Agricultural Statistics. Achal Prakashan Mandir, Kanpur, India, pp. B35±B36. Cheng, M.H., Wang, P.J., Maeda, E., 1987. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explant. Plant Cell Rep. 6, 348±351. Depauw, M.A., Remphrey, W.R., 1993. In vitro germination of three Cypripedium species in relation to time of seed collection, media and cold treatments. Can. J. Bot. 71, 879±885. Fisher, R.A., 1954. Statistical Methods for Research Workers, 12th Edition. Oliver & Boyd, Edinburgh, UK. Fitch, M.M., Mansherdt, R.M., 1990. Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.). Plant Cell Rep. 9, 320±324. Furutani, S.C., Nagao, M.A., 1987. In¯uence of temperature, KNO3 and GA3 and seed drying on emergence of papaya seedlings. Sci. Hort. 32, 67±72.
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Ke, S., Skirvin, R.M., Mcpheeters, K.D., Otterbachet, A.G., Galletta, G., 1985. In vitro germination and growth of Rubus seeds and embryos. Hort. Sci. 20, 1047±1049. Kuwahara, Y.C., Orika, O.E., MarchiLia, D.O.S., 1999. The effect of GA3 on Kiwi (Actinidia chinensis PI) seed germination. Sci. Agric. 56, 9±12. Kyauk, H., Hopper, N.W., Brigham, R.D., 1995. Effect of temperature and presoaking on germination, root length and shoot length of sesame (Sesamum indicum L.). Environ. Exp. Bot. 35, 345±351. Monmarson, S., Micchaux-Ferriere, N., Teisson, C., 1995. Production of high frequency embryogenic calli from integuments of immature seeds of Carica papaya L. J. Hort. Sci. 70, 57±64. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15, 473±497. Murthy, B.N.S., Saxena, P.K., 1998. Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.). Plant Cell Rep. 17, 469±475. Nagao, M.A., Furutani, S.C., 1986. Improving germination of papaya seed by density separation, potassium nitrate, and gibberellic acid. Hort. Sci. 21, 1439±1440. Page, Y.M., Staden, J.Y., 1985. In vitro propagation of Dierama latifolium. Hort. Sci. 20, 1049± 1050. Pancholi, N., Wetten, A., Caligari, P.D.S., 1995. Germination of Musa velutina seeds: comparison of in vivo and in vitro systems. In vitro Cell Dev. Biol. Plantarum 31, 127±130. Sanago, M.M., Murch, S.J., Slimmon, T.Y., Krishnaraj, S., Saxena, P.K., 1995. Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium hortorum Bailey). Plant Cell Rep. 15, 205±211. Sarita, L., Domingos, R.J., 1999. The effect of gibberellins, cytokinins and potassium nitrate on Rangpur lime (Citrus limonia Osbeck) seed germination. Sci. Agric. 56, 111±115. Sen, S.K., Gunthi, P., 1977. Effect of presowing seed treatments on the germination and seedling growth in papaya. Food Farming Agric. 9, 144±146. Shankarraja, N.S., Sulikeri, G.S., 1993. Presowing treatments of seeds to improve germination in cardamom (Elettaria cardamomum L. Maton var minor Watt). J. Plantation Crops 21, 116±117. Tort, N., 1996. Effect of light, different growth media, temperature and salt concentrations on germination of cotton seeds (Gossypium hirsutum L. cv. Nazilli 87). J. Agron. Crop Sci. 176, 217±221. Tseng, T.M., 1991. The effect of GA3 concentration and time of treatment on the promotion of papaya seed germination. Guoli Taiwan Daxue Nongxueyuan Yanjiu Baogao 31, 30±39. Yahiro, M., Yoshitaka, H., 1982. Growth inhibitors in papaya (Carica papaya L.) seeds. Nettai Nogyo. 26, 63±67. Zarad, S.S., Hossny, Y.A., El-Bagoury, H.M., 1997. Effect of various treatments on seed germination and water application on vegetative growth of desert date (Balanite egyptica L.) seedlings grown in different soils. Egypt J. Physiol. Sci. 21, 147±159.