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Micropropagation of Karonda (Carissa carandas) through shoot multiplication
Scientia Horticulturae 103 (2005) 227–232
Short communication
Micropropagation of Karonda (Carissa carandas) through shoot multiplication Ratna Rai, K.K. Misra∗ Department of Horticulture, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, U.S. Nagar, Uttaranchal, India Accepted 16 September 2003
Abstract Shoot tips from field grown, mature plants of Carissa carandas cv. Pant Sudarshan were cultured on Murashige and Skoog’s (MS) basal medium supplemented with benzyladenine (BA) and indolebutyric acid (IBA) during different seasons. The maximum sprouting rate was obtained with 1.5 cm long explant collected in spring season (February–March) followed by those collected in summer season (April–June). Shoot proliferation was highest on MS basal media supplemented with 3.0 mg l−1 BA. Rooting of microshoots was noted to be the best in 1/2 MS plus 0.8 mg l−1 IBA and 0.2 mg l−1 naphthalene acetic acid (NAA). The rooted plantlets were successfully acclimatized in vermiculite:sand:soil (1:1:1) potting mixture. © 2003 Published by Elsevier B.V. Keywords: Benzyladenine; Indolebutyric acid; Naphthalene acetic acid
1. Introduction Karonda (Carissa carandas) popularly known as ‘Christ’s thorn’ is an important minor fruit crop of tropical and subtropical areas and an important crop of arid zones. It has excellent potential to be used for horticultural plantations in marginal and wastelands, owing its hardy nature with wide adaptability to saline sodic soils with pH up to 10 (Bankar et al., 1994; Chundawat, 1995). The fruits possess appreciable amount of jelly grade pectin and acidity, hence a large number of processing factories during the last decade have been built for making commercial jelly and a product by the name ‘Nakal cherry’ which closely resembles the canned cherry fruits (Mandal et al., 1992). Besides, the fruits are one of the richest sources of iron (39.1 mg/100 g). ∗
Corresponding author. Tel.: +91-5944-33363; fax: +91-5944-33473. E-mail address: [email protected] (K.K. Misra). 0304-4238/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.scienta.2003.09.005
228
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
The conventional methods of propagation viz., through seeds, cuttings, grafting, air layering, and stooling (Misra and Jaiswal, 1993; Misra and Singh, 1990; Tyagi et al., 1999) have given variable results which are not very satisfactory. As, C. carandas is widely used for hedge plantations, a large number of plants per unit area are required. Therefore, some alternative method for rapid and large scale multiplication is urgently required in order to increase the area under cultivation of this important underutilized crop. So far, no attempt has been made for micropropagation of Karonda. Therefore, the present investigation was undertaken to study the following aspects of in vitro propagation: (i) regeneration potential of ‘Pant Sudarshan’ an elite cultivar of C. carandas through shoot tip culture; (ii) effect of surface sterilants and their exposure time; (iii) explanting season and length of explant on contamination and sprouting; (iv) levels of plant growth regulators for shoot proliferation; and (v) rooting and hardening media for successful establishment of rooted plantlets.
2. Materials and methods 2.1. Culture conditions All the cultures were grown in a culture room at 25 + 1 ◦ C, 16 h photoperiod with light at 40 mol m2 s−1 (photosynthetic photon flux density) (Phillips “Cool white” fluorescent tubes) at culture level. The vessels used in the experiments were 150 mm×25 mm glass tubes each containing 10 ml of nutrient media and one explant per culture tube was inoculated. The explants were inoculated in MS basal salt formulation adjusted to pH 5.7 and supplemented with 3% sucrose (Sigma Chemical, St. Louis) and 0.7% agar (Sigma Chemical, St. Louis). 2.2. Surface sterilization Shoot tips (3 cm) from the apical part of C. carandas cv. Pant Sudarshan, growing at Horticultural Research Centre, Patharchatta, were collected in five seasons viz., winter (November–January), spring (February–March), summer (April–June), rainy (July–August) and autumn (September–October). The shoot tips were brought from the field in a solution of 1 ml liquid detergent ‘Teepol’ + 0.1% Bavistin + 0.05% streptomycin per 100 ml distilled water. The segments were washed under running tap water for 30 min. The washed segments were then rinsed with double distilled water and brought under laminar airflow hood for further surface sterilization treatments. The shoot tips were sequentially exposed to ethanol (70%), HgCl2 (0.5%) and NaOCl (1%) for different durations. After exposure to surface sterilants, the shoot tips were washed with sterile distilled water (3–4 times) and dried on pre sterilized filter paper sheets. The data on asepsis and sprouting was recorded after a period of 3 weeks. 2.3. Shoot proliferation Shoot tips were trimmed aseptically to 0.5, 1.0 and 1.5 cm long segments and inoculated on MS basal medium supplemented with 0.0, 2.22, 4.44, 8.88, 13.32 or 17.75 M of benzyladenine (BA) and 0.0, 0.88 or 2.28 M of indolebutyric acid (IBA) either alone or in
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
229
various combinations. The pH of the media was adjusted to 5.7. The microshoots that developed on initial explants were further multiplied by subculturing them in fresh medium at 6 weeks interval. Shoot proliferation rate, shoot number and length of shoots was estimated from the means of four subcultures. 2.4. Rooting of microshoots For rooting, shoots longer than 2 cm were isolated from proliferating cultures and transferred to 1/2 MS basal media containing 0.0, 0.57, 1.14, 2.28, 3.42, 4.57 or 5.71 M IBA and 0.0, 1.07 or 2.15 M NAA either alone or in various combinations. Initially the cultures were kept in dark for 7 days. The data on rooting was evaluated after a period of 8 weeks. 2.5. Acclimatization After 30–40 days, the regenerated plantlets with well-developed roots were taken out and washed with sterile distilled water to remove all traces of media. The plantlets were then potted in clean plastic pots filled with different autoclaved potting mixtures viz.: (i) vermiculite; (ii) 1 sand:1 soil:1 farm yard manure (FYM); (iii) 2 sand:1 soil:1 FYM; (iv) 1 sand:2 soil:1 FYM; (v) 1 vermiculite:1 sand:1 soil. The potted plantlets were placed under culture room conditions. Initially for 2 weeks, the plantlets were regularly sprayed with 0.5% Bavistin + 1/8 MS inorganic salts and were covered with inverted glass beakers to ensure high humidity. Subsequently, over a period of 2 weeks, the beakers were gradually removed and the plantlets were adapted to culture room conditions. 2.6. Statistical analysis All the experiments were laid out in completely randomized design (CRD) with 15–20 cultures per replication and each treatment was replicated 10 times. The means were evaluated by applying F-test and critical difference have been calculated at 5% level of significance. Each experiment was repeated twice except the experiments on contamination and sprouting. Data presented in percentages were subjected to arcsine transformation before analysis and then converted back to percentage for presentation.
3. Results and discussion 3.1. Surface sterilization After a period of 3 weeks maximum number of explants survived (74.3%) when they were sequentially exposed to 70% ethanol (30 s), 0.5% HgCl2 (4 min) and 1% NaOCl (3 min). There was minimum contamination in 0.5 cm long explants collected in spring and winter seasons. There was increase in per cent contamination with the increase in length of explant (1.5 cm) and the sprouting frequency (percentage of explants forming buds) was considerably increased with the length of explants. The maximum number of explants sprouted in spring season coinciding with the new vegetative growth of the stock plant.
230
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
3.2. Shoot proliferation Incorporation of various concentrations of BA in the MS basal medium significantly increased the shoot proliferation rate and number and mean length of shoots (Table 1). Increase in BA level to 17.75 M reduced the proliferation rate, number and mean length of shoots. Shoot proliferation and growth decreased considerably with the increase in BAP level in ber (Rathore et al., 1992) and guava (Loh and Rao, 1989). The shoots of Karonda showed vitrification at higher BA concentration (17.75 M). Higher concentration of BA induced vitrification in peach (Chiariotti and Antonelli, 1988). The addition of 0.88 or 2.28 M IBA in the media showed a concentration related decrease in shoot proliferation and number and mean length of shoots in Karonda. The in vitro regenerated shoots were further multiplied by subculturing 1.5 cm long segments individually on MS media containing 13.32 M BA. It was observed that prolifTable 1 Effect of varying levels of plant growth regulators on shoot proliferation and number and mean length of shoots in C. carandasa Plant growth regulators (M)
Shoot proliferation (%)
Number of shoots per explant
Mean length of shoots (cm)
0.00 2.22 4.44 8.88 13.32 17.75
0.00 e 12.41 e 19.47 d 65.17 c 87.52 a 67.39 b
0.00 f 1.48 e 2.90 d 6.60 b 10.13 a 4.66 c
0.00 f 0.48 e 1.22 d 2.08 c 4.39 a 2.38 b
IBA 0.00 0.88 2.28
46.08 a 42.91 b 37.00 c
5.33 a 4.11 b 3.45 c
2.01 a 1.76 b 1.52 c
BA × IBA 0.00 × 0.00 0.00 × 0.88 0.00 × 2.28 2.22 × 0.00 2.22 × 0.88 2.22 × 2.28 4.44 × 0.00 4.44 × 0.88 4.44 × 2.28 8.88 × 0.00 8.88 × 2.28 13.32 × 0.00 13.32 × 0.88 13.32 × 2.28 17.75 × 0.00 17.75 × 0.88 17.75 × 2.28
0.00 m 0.00 m 0.00 m 14.10 k 11.52 l 11.61 l 22.60 h 19.93 i 15.88 j 75.66 d 49.93 g 95.02 a 86.14 b 81.40 c 69.07 e 69.94 e 63.15 f
0.00 o 0.00 o 0.00 o 1.92 l 1.46 m 1.05 n 4.21 i 3.07 k 1.42 m 7.14 d 6.76 e 12.54 a 9.62 b 8.24 c 6.17 f 4.61 h 3.19 j
0.00 o 0.00 o 0.00 o 0.53 m 0.51 m 0.39 n 1.14 l 1.22 k 1.31 j 2.04 g 1.97 h 5.93 a 3.74 b 3.51 c 2.39 e 2.84 d 1.92 i
BA
a
Critical difference at 5% level of significance.
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
231
eration rate and number of shoots increased gradually up to third subculture and thereafter stabilized. Improvement in shoot proliferation rate by increasing number of subcultures in guava has been reported by Singh et al. (2001). 3.3. Rooting of microshoots Shoots (≥2 cm) cultured in 1/2 MS media supplemented with either IBA or NAA alone or in combination showed varying results (Table 2). NAA alone at 1.07 or 2.15 M concentration caused the formation of thick and small roots while IBA alone at all the concentrations Table 2 Effect of varying levels of plant growth regulators on per cent rooting, number and length of roots in C. carandasa Plant growth regulators (M)
Rooting (%)
Number of roots per microshoot
Mean length of roots (cm)
IBA 0.00 0.57 1.14 2.28 3.42 4.57 5.71
3.20 g 6.63 f 19.52 e 31.77 d 42.49 c 69.11 a 60.19 b
0.70 g 2.55 f 4.61 e 6.07 c 5.69 d 10.66 a 6.98 b
0.30 g 1.26 f 1.78 e 3.42 b 3.02 c 4.42 a 2.30 d
NAA 0.00 1.07 2.15
33.73 b 37.10 a 28.98 c
4.55 b 6.56 a 4.79 b
2.19 b 2.90 a 1.91 c
IBA × NAA 0.00 × 0.00 0.00 × 1.07 0.00 × 2.15 0.57 × 0.00 0.57 × 1.07 0.57 × 2.15 1.14 × 0.00 1.14 × 1.07 1.14 × 2.15 2.28 × 0.00 2.28 × 1.07 2.28 × 2.15 3.42 × 0.00 3.42 × 1.07 3.42 × 2.15 4.57 × 0.00 4.57 × 1.07 4.57 × 2.15 5.71 × 0.00 5.71 × 1.07 5.71 × 2.15
0.00 t 6.00 q 3.60 s 5.03 r 7.33 p 7.53 p 28.47 l 20.30 n 9.80 o 36.70 j 27.83 m 30.77 k 41.20 h 46.80 g 39.47 i 71.90 b 81.33 a 54.10 e 52.80 f 70.13 c 57.63 d
0.00 o 1.10 n 1.00 n 1.60 m 3.53 k 2.53 l 3.97 j 5.27 h 4.60 i 5.30 h 7.20 e 5.70 h 4.57 i 6.60 f 5.40 h 10.33 b 12.83 a 8.80 d 6.07 g 9.37 c 5.50 h
0.00 j 0.40 i 0.50 i 0.87 h 1.27 g 1.67 g 1.17 g 2.33 f 1.83 g 3.43 d 4.10 c 2.73 e 3.50 d 3.67 d 1.40 g 4.57 b 5.40 a 3.30 d 1.83 g 3.13 d 1.93 g
a
Critical difference at 5% level of significance.
232
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
tested formed thin and long roots. Better rooting was observed when both the auxins were used in combination. The use of two auxins for better rooting has also been suggested by earlier workers (Amin and Jaiswal, 1987; Kornova, 1995). Initial incubation of cultures for 7 days in dark advanced rooting. Once the initiation of roots took place in the dark, the cultures were shifted to light which resulted in normal growth of roots. 3.4. Acclimatization Among all the potting mixtures, better establishment of plantlets was observed in vermiculite:sand:soil (1:1:1) with a survival frequency of 80.70%.
References Amin, M.N., Jaiswal, V.S., 1987. Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature tree. Plant Cell Tiss. Org. Cult. 9, 235–243. Bankar, G.J., Verma, S.K., Prasad, R.N., 1994. Fruit for the arid region: Karonda. Indian Hort. 39 (1), 46–47. Chiariotti, A., Antonelli, M., 1988. The effect of 6-BAP and adenine sulphate on peach shoot proliferation. Acta Hort. 227, 418–420. Chundawat, B.S., 1995. Arid Fruit Culture. Oxford and IBH Publishing Company Private Limited, New Delhi. Kornova, K., 1995. Study of in vitro rooting in some peach varieties “Rasteniev”. Dni-Nauki 32 (7–8), 109–111. Loh, C.S., Rao, A.N., 1989. Clonal propagation of guava (Psidium guajava L.) from seedling and grafted plants and adventitious shoot formation in vitro. Sci. Hort. 39, 31–39. Mandal, U., Sinha Roy, S., Mazumdar, B.C., 1992. A recently developed agroindustry in the southern suberb of Calcutta city, utilizing a bramble fruit. Indian J. Landsc. Syst. Ecol. Stud. 15 (1), 100–102. Misra, K.K., Jaiswal, H.R., 1993. Effect of growth regulators on rooting of stool layers of Karonda (Carissa carandas L.). Indian J. For. 16 (2), 181–182. Misra, K.K., Singh, R., 1990. Effect of growth regulators on rooting and survival of air layers of Karonda (Carissa carandas L.). Ann. Agric. Res. 11 (2), 208–210. Rathore, T.S., Singh, R.P., Deora, N.S., Shekhawat, N.S., 1992. Clonal propagation of Ziziphus species through tissue culture. Sci. Hort. 51, 165–168. Singh, S.K., Singh, S.P., Sharma, H.C., 2001. In vitro clonal propagation of guava (P. guajava L.) from field grown mature plants. Physiol. Mol. Biol. Plants 7, 33–38. Tyagi, S., Misra, K.K., Jaiswal, H.R., 1999. Effect of auxins on rooting of softwood stem cuttings of Carissa under mist. Sci. Hort. 6, 37–43.
Short communication
Micropropagation of Karonda (Carissa carandas) through shoot multiplication Ratna Rai, K.K. Misra∗ Department of Horticulture, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, U.S. Nagar, Uttaranchal, India Accepted 16 September 2003
Abstract Shoot tips from field grown, mature plants of Carissa carandas cv. Pant Sudarshan were cultured on Murashige and Skoog’s (MS) basal medium supplemented with benzyladenine (BA) and indolebutyric acid (IBA) during different seasons. The maximum sprouting rate was obtained with 1.5 cm long explant collected in spring season (February–March) followed by those collected in summer season (April–June). Shoot proliferation was highest on MS basal media supplemented with 3.0 mg l−1 BA. Rooting of microshoots was noted to be the best in 1/2 MS plus 0.8 mg l−1 IBA and 0.2 mg l−1 naphthalene acetic acid (NAA). The rooted plantlets were successfully acclimatized in vermiculite:sand:soil (1:1:1) potting mixture. © 2003 Published by Elsevier B.V. Keywords: Benzyladenine; Indolebutyric acid; Naphthalene acetic acid
1. Introduction Karonda (Carissa carandas) popularly known as ‘Christ’s thorn’ is an important minor fruit crop of tropical and subtropical areas and an important crop of arid zones. It has excellent potential to be used for horticultural plantations in marginal and wastelands, owing its hardy nature with wide adaptability to saline sodic soils with pH up to 10 (Bankar et al., 1994; Chundawat, 1995). The fruits possess appreciable amount of jelly grade pectin and acidity, hence a large number of processing factories during the last decade have been built for making commercial jelly and a product by the name ‘Nakal cherry’ which closely resembles the canned cherry fruits (Mandal et al., 1992). Besides, the fruits are one of the richest sources of iron (39.1 mg/100 g). ∗
Corresponding author. Tel.: +91-5944-33363; fax: +91-5944-33473. E-mail address: [email protected] (K.K. Misra). 0304-4238/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.scienta.2003.09.005
228
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
The conventional methods of propagation viz., through seeds, cuttings, grafting, air layering, and stooling (Misra and Jaiswal, 1993; Misra and Singh, 1990; Tyagi et al., 1999) have given variable results which are not very satisfactory. As, C. carandas is widely used for hedge plantations, a large number of plants per unit area are required. Therefore, some alternative method for rapid and large scale multiplication is urgently required in order to increase the area under cultivation of this important underutilized crop. So far, no attempt has been made for micropropagation of Karonda. Therefore, the present investigation was undertaken to study the following aspects of in vitro propagation: (i) regeneration potential of ‘Pant Sudarshan’ an elite cultivar of C. carandas through shoot tip culture; (ii) effect of surface sterilants and their exposure time; (iii) explanting season and length of explant on contamination and sprouting; (iv) levels of plant growth regulators for shoot proliferation; and (v) rooting and hardening media for successful establishment of rooted plantlets.
2. Materials and methods 2.1. Culture conditions All the cultures were grown in a culture room at 25 + 1 ◦ C, 16 h photoperiod with light at 40 mol m2 s−1 (photosynthetic photon flux density) (Phillips “Cool white” fluorescent tubes) at culture level. The vessels used in the experiments were 150 mm×25 mm glass tubes each containing 10 ml of nutrient media and one explant per culture tube was inoculated. The explants were inoculated in MS basal salt formulation adjusted to pH 5.7 and supplemented with 3% sucrose (Sigma Chemical, St. Louis) and 0.7% agar (Sigma Chemical, St. Louis). 2.2. Surface sterilization Shoot tips (3 cm) from the apical part of C. carandas cv. Pant Sudarshan, growing at Horticultural Research Centre, Patharchatta, were collected in five seasons viz., winter (November–January), spring (February–March), summer (April–June), rainy (July–August) and autumn (September–October). The shoot tips were brought from the field in a solution of 1 ml liquid detergent ‘Teepol’ + 0.1% Bavistin + 0.05% streptomycin per 100 ml distilled water. The segments were washed under running tap water for 30 min. The washed segments were then rinsed with double distilled water and brought under laminar airflow hood for further surface sterilization treatments. The shoot tips were sequentially exposed to ethanol (70%), HgCl2 (0.5%) and NaOCl (1%) for different durations. After exposure to surface sterilants, the shoot tips were washed with sterile distilled water (3–4 times) and dried on pre sterilized filter paper sheets. The data on asepsis and sprouting was recorded after a period of 3 weeks. 2.3. Shoot proliferation Shoot tips were trimmed aseptically to 0.5, 1.0 and 1.5 cm long segments and inoculated on MS basal medium supplemented with 0.0, 2.22, 4.44, 8.88, 13.32 or 17.75 M of benzyladenine (BA) and 0.0, 0.88 or 2.28 M of indolebutyric acid (IBA) either alone or in
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
229
various combinations. The pH of the media was adjusted to 5.7. The microshoots that developed on initial explants were further multiplied by subculturing them in fresh medium at 6 weeks interval. Shoot proliferation rate, shoot number and length of shoots was estimated from the means of four subcultures. 2.4. Rooting of microshoots For rooting, shoots longer than 2 cm were isolated from proliferating cultures and transferred to 1/2 MS basal media containing 0.0, 0.57, 1.14, 2.28, 3.42, 4.57 or 5.71 M IBA and 0.0, 1.07 or 2.15 M NAA either alone or in various combinations. Initially the cultures were kept in dark for 7 days. The data on rooting was evaluated after a period of 8 weeks. 2.5. Acclimatization After 30–40 days, the regenerated plantlets with well-developed roots were taken out and washed with sterile distilled water to remove all traces of media. The plantlets were then potted in clean plastic pots filled with different autoclaved potting mixtures viz.: (i) vermiculite; (ii) 1 sand:1 soil:1 farm yard manure (FYM); (iii) 2 sand:1 soil:1 FYM; (iv) 1 sand:2 soil:1 FYM; (v) 1 vermiculite:1 sand:1 soil. The potted plantlets were placed under culture room conditions. Initially for 2 weeks, the plantlets were regularly sprayed with 0.5% Bavistin + 1/8 MS inorganic salts and were covered with inverted glass beakers to ensure high humidity. Subsequently, over a period of 2 weeks, the beakers were gradually removed and the plantlets were adapted to culture room conditions. 2.6. Statistical analysis All the experiments were laid out in completely randomized design (CRD) with 15–20 cultures per replication and each treatment was replicated 10 times. The means were evaluated by applying F-test and critical difference have been calculated at 5% level of significance. Each experiment was repeated twice except the experiments on contamination and sprouting. Data presented in percentages were subjected to arcsine transformation before analysis and then converted back to percentage for presentation.
3. Results and discussion 3.1. Surface sterilization After a period of 3 weeks maximum number of explants survived (74.3%) when they were sequentially exposed to 70% ethanol (30 s), 0.5% HgCl2 (4 min) and 1% NaOCl (3 min). There was minimum contamination in 0.5 cm long explants collected in spring and winter seasons. There was increase in per cent contamination with the increase in length of explant (1.5 cm) and the sprouting frequency (percentage of explants forming buds) was considerably increased with the length of explants. The maximum number of explants sprouted in spring season coinciding with the new vegetative growth of the stock plant.
230
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
3.2. Shoot proliferation Incorporation of various concentrations of BA in the MS basal medium significantly increased the shoot proliferation rate and number and mean length of shoots (Table 1). Increase in BA level to 17.75 M reduced the proliferation rate, number and mean length of shoots. Shoot proliferation and growth decreased considerably with the increase in BAP level in ber (Rathore et al., 1992) and guava (Loh and Rao, 1989). The shoots of Karonda showed vitrification at higher BA concentration (17.75 M). Higher concentration of BA induced vitrification in peach (Chiariotti and Antonelli, 1988). The addition of 0.88 or 2.28 M IBA in the media showed a concentration related decrease in shoot proliferation and number and mean length of shoots in Karonda. The in vitro regenerated shoots were further multiplied by subculturing 1.5 cm long segments individually on MS media containing 13.32 M BA. It was observed that prolifTable 1 Effect of varying levels of plant growth regulators on shoot proliferation and number and mean length of shoots in C. carandasa Plant growth regulators (M)
Shoot proliferation (%)
Number of shoots per explant
Mean length of shoots (cm)
0.00 2.22 4.44 8.88 13.32 17.75
0.00 e 12.41 e 19.47 d 65.17 c 87.52 a 67.39 b
0.00 f 1.48 e 2.90 d 6.60 b 10.13 a 4.66 c
0.00 f 0.48 e 1.22 d 2.08 c 4.39 a 2.38 b
IBA 0.00 0.88 2.28
46.08 a 42.91 b 37.00 c
5.33 a 4.11 b 3.45 c
2.01 a 1.76 b 1.52 c
BA × IBA 0.00 × 0.00 0.00 × 0.88 0.00 × 2.28 2.22 × 0.00 2.22 × 0.88 2.22 × 2.28 4.44 × 0.00 4.44 × 0.88 4.44 × 2.28 8.88 × 0.00 8.88 × 2.28 13.32 × 0.00 13.32 × 0.88 13.32 × 2.28 17.75 × 0.00 17.75 × 0.88 17.75 × 2.28
0.00 m 0.00 m 0.00 m 14.10 k 11.52 l 11.61 l 22.60 h 19.93 i 15.88 j 75.66 d 49.93 g 95.02 a 86.14 b 81.40 c 69.07 e 69.94 e 63.15 f
0.00 o 0.00 o 0.00 o 1.92 l 1.46 m 1.05 n 4.21 i 3.07 k 1.42 m 7.14 d 6.76 e 12.54 a 9.62 b 8.24 c 6.17 f 4.61 h 3.19 j
0.00 o 0.00 o 0.00 o 0.53 m 0.51 m 0.39 n 1.14 l 1.22 k 1.31 j 2.04 g 1.97 h 5.93 a 3.74 b 3.51 c 2.39 e 2.84 d 1.92 i
BA
a
Critical difference at 5% level of significance.
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
231
eration rate and number of shoots increased gradually up to third subculture and thereafter stabilized. Improvement in shoot proliferation rate by increasing number of subcultures in guava has been reported by Singh et al. (2001). 3.3. Rooting of microshoots Shoots (≥2 cm) cultured in 1/2 MS media supplemented with either IBA or NAA alone or in combination showed varying results (Table 2). NAA alone at 1.07 or 2.15 M concentration caused the formation of thick and small roots while IBA alone at all the concentrations Table 2 Effect of varying levels of plant growth regulators on per cent rooting, number and length of roots in C. carandasa Plant growth regulators (M)
Rooting (%)
Number of roots per microshoot
Mean length of roots (cm)
IBA 0.00 0.57 1.14 2.28 3.42 4.57 5.71
3.20 g 6.63 f 19.52 e 31.77 d 42.49 c 69.11 a 60.19 b
0.70 g 2.55 f 4.61 e 6.07 c 5.69 d 10.66 a 6.98 b
0.30 g 1.26 f 1.78 e 3.42 b 3.02 c 4.42 a 2.30 d
NAA 0.00 1.07 2.15
33.73 b 37.10 a 28.98 c
4.55 b 6.56 a 4.79 b
2.19 b 2.90 a 1.91 c
IBA × NAA 0.00 × 0.00 0.00 × 1.07 0.00 × 2.15 0.57 × 0.00 0.57 × 1.07 0.57 × 2.15 1.14 × 0.00 1.14 × 1.07 1.14 × 2.15 2.28 × 0.00 2.28 × 1.07 2.28 × 2.15 3.42 × 0.00 3.42 × 1.07 3.42 × 2.15 4.57 × 0.00 4.57 × 1.07 4.57 × 2.15 5.71 × 0.00 5.71 × 1.07 5.71 × 2.15
0.00 t 6.00 q 3.60 s 5.03 r 7.33 p 7.53 p 28.47 l 20.30 n 9.80 o 36.70 j 27.83 m 30.77 k 41.20 h 46.80 g 39.47 i 71.90 b 81.33 a 54.10 e 52.80 f 70.13 c 57.63 d
0.00 o 1.10 n 1.00 n 1.60 m 3.53 k 2.53 l 3.97 j 5.27 h 4.60 i 5.30 h 7.20 e 5.70 h 4.57 i 6.60 f 5.40 h 10.33 b 12.83 a 8.80 d 6.07 g 9.37 c 5.50 h
0.00 j 0.40 i 0.50 i 0.87 h 1.27 g 1.67 g 1.17 g 2.33 f 1.83 g 3.43 d 4.10 c 2.73 e 3.50 d 3.67 d 1.40 g 4.57 b 5.40 a 3.30 d 1.83 g 3.13 d 1.93 g
a
Critical difference at 5% level of significance.
232
R. Rai, K.K. Misra / Scientia Horticulturae 103 (2005) 227–232
tested formed thin and long roots. Better rooting was observed when both the auxins were used in combination. The use of two auxins for better rooting has also been suggested by earlier workers (Amin and Jaiswal, 1987; Kornova, 1995). Initial incubation of cultures for 7 days in dark advanced rooting. Once the initiation of roots took place in the dark, the cultures were shifted to light which resulted in normal growth of roots. 3.4. Acclimatization Among all the potting mixtures, better establishment of plantlets was observed in vermiculite:sand:soil (1:1:1) with a survival frequency of 80.70%.
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