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Growth regulators effect on callus induction J. Agric. Res., 2015, 53(2) 233 EFFECT OF GROWTH REGULATORS ON CALLUS INDUCTION AND MICROPROPAGATION OF THREE GRAPE VARIETIES Neelma Munir, Ifrah Safdar and Shagufta Naz* ABSTRACT A study was conducted in the Department of Biotechnology, Lahore College for Women University Lahore, Pakistan during 2012 to optimize the protocol for callus induction and micro propagation of grapes varieties i.e. Desi (V1), Sundar Khani (V2) and Chinese grapes (V3). Nodal leaves were used as explants for callus indication. Sodium hydrochloride was used for sterilization of explants. Though there was difference in the survival percentage, the best response was obtained when the explants were treated with 5 percent sodium hypochlorite for 10 minutes. Early callus formation was obtained in MS (Murashige Skoog) medium supplemented with 2,4-D at the concentration of 3.0mg per liter in 22 days. The highest value of shoot length was observed in MS medium supplemented with 1mg per liter BAP. In V1 the mean shoot length observed after 18.6±7.122days was 4.2±1.73cm whereas in V2 and V3 it was 4.5±1.79 and 4.0 ±1.63cm respectively. Best rooting was achieved on MS media supplemented with 1mg per liter NAA. KEYWORDS: Grapes cultivars; callus induction; plant growth regulators; Pakistan. INTRODUCTION Grape is one of the oldest and best vital perennial crop in the world (4).It is effectively cultivated only in stable and temperate climate regions with plenty of rainfall, warm and dry summers and comparatively mild winters (17). In Pakistan, grapes are cultivated on an area of 13,000 hectare and annual production of 49.0 thousand tons (2). Grapes are very nutritious and contains 288 KJ (69 kcal) of energy per 100 g (5). It contains 18.1g carbohydrates, 15.48g sugars, 0.9g dietary fiber, 0.72 g protein per 100g of the sample. Dietary value of grapes reveals different proportions of vitamins (B1, B2, B3, B5, B6, B9, B12) calcium, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc that also flavors the nutritional importance of *Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan.

EFFECT OF GROWTH REGULATORS ON CALLUS ......Growth regulators effect on callus induction J. Agric. Res., 2015, 53(2) 235 Inoculation: After sterilization, and media formulation midrib

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Growth regulators effect on callus induction

J. Agric. Res., 2015, 53(2)

233

EFFECT OF GROWTH REGULATORS ON CALLUS INDUCTION AND MICROPROPAGATION OF THREE

GRAPE VARIETIES

Neelma Munir, Ifrah Safdar and Shagufta Naz*

ABSTRACT

A study was conducted in the Department of Biotechnology, Lahore College for Women University Lahore, Pakistan during 2012 to optimize the protocol for callus induction and micro propagation of grapes varieties i.e. Desi (V1), Sundar Khani (V2) and Chinese grapes (V3). Nodal leaves were used as explants for callus indication. Sodium hydrochloride was used for sterilization of explants. Though there was difference in the survival percentage, the best response was obtained when the explants were treated with 5 percent sodium hypochlorite for 10 minutes. Early callus formation was obtained in MS (Murashige Skoog) medium supplemented with 2,4-D at the concentration of 3.0mg per liter in 22 days. The highest value of shoot length was observed in MS medium supplemented with 1mg per liter BAP. In V1 the mean shoot length observed after 18.6±7.122days was 4.2±1.73cm whereas in V2 and V3 it was 4.5±1.79 and 4.0 ±1.63cm respectively. Best rooting was achieved on MS media supplemented with 1mg per liter NAA.

KEYWORDS: Grapes cultivars; callus induction; plant growth regulators; Pakistan.

INTRODUCTION

[Grape is one of the oldest and best vital perennial crop in the world (4).It is effectively cultivated only in stable and temperate climate regions with plenty of rainfall, warm and dry summers and comparatively mild winters (17). In Pakistan, grapes are cultivated on an area of 13,000 hectare and annual production of 49.0 thousand tons (2). Grapes are very nutritious and contains 288 KJ (69 kcal) of energy per 100 g (5). It contains 18.1g carbohydrates, 15.48g sugars, 0.9g dietary fiber, 0.72 g protein per 100g of the sample. Dietary value of grapes reveals different proportions of vitamins (B1, B2, B3, B5, B6, B9, B12) calcium, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc that also flavors the nutritional importance of

*Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan.

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grapes (12). Grapes comprises multipart compounds such as sugars in the form of glucose and fructose, acids like tartaric and malic, amino acids, proteins, anthocyanin and flavonals, but apart from these the abundant amount of water is present (22). As grape is a heterozygous and vegetatively propagated fruit crop. It needs lengthy period to sprout flowering due to extensive juvenile stage(11).Clonal selection and breeding are routinely used for crop improvement of grapes so, for the development of fruit by conventional methods is quite time taking (13, 18). Plant tissue culture has provided the standards of success and sensation to the nation‘s economic condition. This scientific technique has always been helpful in maintaining and sustaining plants through eradicating microbes and drastic diseases, by securing endangered species and by the production of desired varieties in lesser span of time. The callus induction and micropopagation of grapes is well documented in literature (6) but studies with cultivars of V. vinifera L., have met with limited success (3, 21). The grape cultivars used during the present work have not been exploited yet and for any further work on these it is a prerequisite to establish protocols for in vitro culture.

MATERIALS AND METHODS

This present study was conducted in Department of Biotechnology, Lahore college for Women University Lahore, Pakistan during 2012. Three selected grape varieties Desi (V1), Sundar Khani (V2) and Chinese grapes (V3) were used in the experiment. Nodel portion and leaves of young branches were used as explant. Aseptic techniques: Standard aseptic techniques used in plant tissue minimize culture were applied in order to minimize the chances of contamination. Explants were surface sterilized with 5 % sodium hypochlorite solution with a few drops of tween 20. The UV lamp of laminar air flow cabinet was switched on half an hour before inoculation Media Preparation: For preparation of MS medium stock solutions of the macronutrients, micronutrients, vitamins, iron and growth regulators were prepared. Analytical grade chemicals and double distilled water was used to prepare all the stock solutions. MS medium (9) was supplemented with various concentrations of growth hormones to standardize the nutritional requirements for callus induction and micro-propagation.

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Inoculation: After sterilization, and media formulation midrib of the leaf was cut into pieces of 1cm size. Nodal explants of about 1.5 cm were used for inoculation. The explants were then inoculated on Murashige and Skoog’s (MS) medium supplemented with different concentrations of growth regulators. During the present work the concentration of 2,4-D, benzyl adenine, naphthalene acetic acid and kinetin was optimized to get better response of callus induction as well as micropropagation. . The cultured explants were observed on daily basis after inoculation and the contaminated test tubes were discarded. Percentage of contamination, callus formation, micropropagation and frequency of proliferated explants was recorded at 30th day. Culture Conditions: For all the experiments cultures were kept at 25°C under 16-hours photoperiod (35 mmol/ m2/ s1) provided by cool fluorescent tube lights.

RESULTS AND DISCUSSION

Standerdization of sterlization process for different explants: With the aim of creating the maximum contaminant free cultures, leaves and nodes dissected from the grapes were treated with various concentrations of sodium hypochlorite (NaOCl) for variable durations of time (Table 1). Table 1. Effect of surface disinfectants on contamination percentage and number of contamination free cultures established.

Sr. No.

Percentage of sodium hypochlorite used (%)

Exposure time (min)

Survival of leaf explants (%)

Survival of nodal explants (%)

V1 V2 V3 V1 V2 V3 1. 5 5 80% 80% 80% 60% 80% 80% 2. 5 10 100% 100% 95% 100% 100% 100% 3. 5 15 10% 30% 10% 70% 50% 10% 4. 10 5 40% 50% 40% 60% 40% 40% 5. 10 10 10% 20% 10% 20% 20% 10% 6. 10 15 0% 0% 0% 0% 0% 0%

It is evident from the table that 100 percent of contaminant free leaf and nodal explants of V1and V2were established when they were exposed to 5 percent sodium hypochlorite for the duration of 10 minutes. For V3 the same combination of treatment was found to be effective with 95 percent survival of explants. Higher concentration of the sterilizing agent or increasing the duration resulted in browning of the leaf explants.

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It has also been found sodium hypochlorite to be an effective sterilizing agent for grapes (3) however the use of other sterilizing agents like chlorox has also been reported (7, 20) but with less percentage (41.3%) of clean cultures. Standerdization of growth regulators for callus growth: The MS medium supplemented with various growth regulators were selected and tried for callus initiation of Vitis vinifera L. It was perceived that the callus formation depends upon the explant used, nature, concentrations of hormones and culture conditions. Callus induction from leaf explants: Leaf as an explant was inoculated on MS basal medium supplemented with diverse concentrations of 2, 4-D (mg/L). Early callus formation was perceived in MS medium supplemented with 2, 4-D 3.0 mg/L in 22 days whereas maximum time noted for callus induction was more than 2 months with 2,4-D (2.0 mg/L). It is evident from Table 2 that 2 mg/L 2,4-D gave effective response of callus induction (50, 60 and 45% V1,V2 and V3 respectively) in 40 days also shown in Fig 1.

Fig 1. Callus formation from leaf explant in MSC1 for V1 and V3 (Age 20 days)

At 3mg/L 2, 4-D good proliferated callus was obtained. At this concentration rate of callus induction was 90 percent in V1, 95 percent in V2 and 80 percent in V3. By increasing the concentration of 2, 4-D to 4mg/L the rate of callus induction was decreased and 75, 80 percent and 70 percent callus induction was observed in grape variety V1, V2 and V3respectively. When 3.0 mg/L 2, 4-D was supplemented with different concentrations of BAP (1.0, 1.5 and 2.0 mg/L) a slight change was noticed. At 1.5 mg/L BAP maximum callus induction was recorded in V2 (85%) in 25 days (Fig 2).

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Fig 2. Callus formed in MSC 8 from leaf explant at day 27 for variety a) V1 b) V2 and c) V3

It has also been reported that the color and texture of callus was greenish white in the start but grew rapidly into yellowish brown nodular friable callus on subsequent culture (20). During the present work different combinations of 2,4-D and kinetin were tested for callus induction. The best response was observed in MSC8 (2,4-D 5 mg/L + 2.5 mg/L kin) which produces 70% callus induction in V1, whereas in V2 and V3 it was 75 and 65 percent, respectively. Callus induction from nodal explants: Nodes as an explant was inoculated on MS basal medium supplemented with various growth regulators. The results are given in Table 3. Good response was observed with 3 mg/L 2,4-D and 1mg/L BAP (Fig 3). Frequency of callus at this concentration was 85 percent in V1, 90 percent in V2 while V3 showed 80 percent response. But best response was recorded with BAP 0.25mg/L + 1mg/L NAA where nodal segments induced 90 percent callus in V1, 95 percent in V2 and 80 percent in V3. Benzyl amino purine (BAP) supplemented with NAA proved to be the best media in enhancing callus weight by nodal explants.

Fig 3. Callus formation from nodal explants in MSC 3 for V2 and V3 (Age 40 days)

In a study MS medium supplemented with different concentrations of BAP (1mg/L) and 2,4-D (1mg/L) was used and found that growth hormones are

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very important for callus formation of all grape cultivars (1). Callus induction has also been reported in MS medium supplemented with various combinations of 2,4-D and BAP by many earlier workers (8, 15, 19).

Standerdization of growth regulators for shoot growth: Different growth regulators were used for micropropagation of shoot of Vitis vinifera L.Nodes as an explant was inoculated on MS basal medium supplemented with various concentrations of BAP (mg/L). It is evident from Table 4 that MS medium having1 mg/L BAP considerably affected on shoot proliferation and good plant height was observed among cultures. At this concentration shoot induction was observed in 85% of cultures in V1, 90% in V2 and 80% in V3. Maximum shoot length observed after 15 ± 7.98 days in the three tested grape varieties was 4.2±1.73, 4.5±1.79 and 4.0±1.63cm respectively (Fig. 4).

Fig. 4. Shoot formation on MS medium supplemented 2.5 mg/L BAP+3.0 mgL-1 TDZ with in variety a) V1at day 18 b) V2 at day 17 c) V3 at day 20.

It is evident from the Table that when NAA was supplemented with different concentrations of BAP (3.0, 2.0 and 1.0mg/L) no significant response of shoot length was observed.

Various concentrations of TDZ (1.0, 2.5, and 3.0mg/L) were tested in combination with 2 mg/L BAP. The earliest response was recorded in MS 9 medium (BAP 2.5 mg/L + 3mg/L TDZ) where shoot induction was observed in 18±7.107days in V1. V2 used during the present work gave quicker response amongst all the three varieties after 17±7.101 days.

The highest value of shoot length was 3.8±1.50 cm in V2 whereas in V1 and V3 it was 3.5±1.32cm with MS9 medium (2.5 mg/L BAP + TDZ 3.0mg/L). The maximum time required for shoot initiation was (35 days) with (2.5 mg/L BAP + 1mg/L TDZ ) with shoot length of 2.4±0.68 cm in V3 while V1 and V2 had shoot length of 2.9±0.98 cm in 32 and 30 days respectively (Table 4).

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Jaskani et al. (7) found that BAP mg/L produced high shoot proliferation (7). Likewise, BA (5 µM) supplemented MS medium was considered as a best media for shoot formation (12). In another study, 2 mg/L BAP was found to be a promising media for full establishment of axillary buds (17).

Fig. 5. Root induction in MS medium for V2 and V3 on MS medium supplemented with NAA.

It was observed that TDZ was not very effective growth regulator as far as shoot induction response of grape varieties is concerned. Though an increase was observed in shoot induction response in less time but all the combinations did not give better response as compared to BAP at the concentration of 1mg/L. It has also been found that different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ) give less response as compared to 2,4-D with 6-benzyl amino purine (10).

ROOTING Well regenerated shoots were shifted to root induction medium. NAA 1mg/L was considered as the best medium and good rooting response was observed (Table 5).The highest (80%) response was recorded in MS medium supplemented with 1mg/L NAA.

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Table 5. Influence of growth regulators on root induction from nodal explant in grapes.

Each value is mean of twenty replicates with standard error (mean ± S.E). Mean along with different letters in the same column vary significantly at p =0.05 according to Duncan’s new multiple range test.

REFERENCES

1. Bayir, A., H. I. Uzun and A. Yalcin. 2007. Effect of genotype on callus formation and organogenesis in Vitis. Acta Hortic. 754:111-116.

2. Bohm, A. and E. Zyprian. 1998. RAPD marker in grapevine (Vitis spp.) similar to plant retrotransposons. Plant Cell Rep.17:415-421.

3. Chee, R. and R. M. Pool. 1985. In vitro propagation of Vitis: the effects of organic substances on shoot multiplication. Vitis. 24:106-118.

4. Das, B. K., R.C Jena and K.C Samal. 2005. Optimization of DNA isolation and PCR protocol for RAPD analysis of banana / plantain (Musa spp.). Int. J. Agri. Sci. 1:21-25.

5. Einset, J. and C. Pratt. 1975. Grapes, in Advances in Fruit Breeding. Moore J. N. and Janick, J. (eds.), Purdue University Press, Lafayette, Indiana. p. 130-153.

6. Gray, D. J. and L. C. Fischer. 1985. In vitro shoot propagation of grape species, hybrids and cultivars. Proc. Fla. State Hort. Soc. 98:172-174.

7. Jaskani, M. J., H. Abbas, R. Sultana, M. M. Khan, M. Qasim and I. A. Khan. 2008. Effect of growth hormones on micropropagation of Vitis vinifera L. CV. Perlette. Pak. J. Bot., 40(1): 105-109.

8. Krastanova, S., K. S. Ling, H. Y. Zhu, B. Xue, T. J. Burr and D. Gonsalves. 2000. Development of transgenic grapevine rootstocks with genes from grapevine fan leaf virus and grapevine leaf roll associated closteroviruses 2 and 3. Acta Hortic. 528: 367-372.

Medium

Growth regulator

Conc. (mg/L)

Frequency of roots (%)

Mean No. of roots

V1 R1 R2 R3

MS+NAA

1.0 2.0 3.0

60% 50% 45%

5±1.79bc

3±1.04cd 2±0.47d

V2 R1 R2 R3

MS+NAA

1.0 2.0 3.0

80% 70% 50%

7±2.81a 5±1.80b 3±1.09c

V3 R1 R2 R3

MS+NAA

1.0 2.0 3.0

70% 65% 40%

2±0.41de 1±0.24e 1±0.21f

LSD 9.6

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9. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15(3):473-497.

10. Olah, R., A. Zoka, A. Pedryca, S. Howardb and L. G. Kovacsb. 2009. Somatic embryogenesis in a broad spectrum of grape genotypes. Sci. Hort. 120(1):134-137.

11. Olmo, H. P. 1942. Choice of parents as influencing seed germination in fruits. Proc. Amer. Soc. Hort. Sci.41: 171-175.

12. Papyia, O. E. E., A. A. Rizkalla and S. R. S. Sabri. 2009. In vitro mutagenesis for genetic improvement of salinity tolerance in wheat. Res.J. Agric. Biol. Sci. 4(5):377-383.

13. Reisch, B. I., D. V. Peterson and M. H. Martens. 2002. Wine phenolics. Ann. N. Y. Acad. Sci. 957:21-36.

14. Sajid, G. M., S. D. Siddique, M. Ishtiaq, A.U. Haq and A. Rashid. 2003. In vitro conservation of grapes. Pak. J. Bot. 38(1): 85-91.

15. Salunkhe, C. K., P. S. Rao and M. Mhatre. 1999. Plantlet regeneration via somatic embryogenesis in anther callus of Vitislatifolia L. Plant Cell Res. 18(7):670-673.

16. Sato, M., G. A. Cordis., N. Maulik and D. K. Das. 2000. SAPKs regulation of ischemic preconditioning. Amer. J. Phys. 27(9):901-907.

17. Shinde., K. A and R. M. Patel. 2009. Proliferation, rooting and acclimatization of micropropagated grape cv. Thompson seedless. Inter. J. Plant. Sci. 5(1):98-101.

18. Vivier, M. A and I. S. Pretorius. 2000. Genetic improvement of grapevine: tailoring grape varieties for the third millennium. S. Afr. J .Enol. Vitic. 21:5-26.

19. Wang,C. T., Y. Y. Tang, X. Z. Wang, S.W. Zhang, G. J. Li, J. C. Zhang and S. L. Yu. 2010. Sodium azide mutagenesis resulted in a peanut plant with elevated oleate content. Elec. J. Biotech. 14(2):1-7.

20. Xu, X., J. Lu, Z. Ren, H. Wang and S. Leong. 2005. Callus induction and somatic embryogenesis in muscadine and seedless bunch grapes (Vitis) from immature ovule culture. Proc. Fla. State. Hort. Soc. 118: 260-262.

21. Zatiko, J. M. and I. Molnar. 1985. Preliminary results on the in vitro mass propagation of grapes from shoot-tip meristem. Fruit Sci. Rep.12:83-85.

22. Zhang, Q. R., J. Z. Sun, N. H. Ren, X. Y. Dong, Z. M. Liu and M. Zhai. 2006. Tissue culture of grapes. J. Agri. Sci. 24:88-90.

Received: October 8, 2013 Accepted: October 15, 2014

***

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Table 2. Influence of growth hormones on callus in V1, V2 and V3 from leaf explant.

Each value is mean of twenty replicates with standard error (mean ± S.E). Mean along with different letters in the same column vary significantly at P =0.05 according to Duncan’s new multiple range test.

Medium

Growth regulator

Conc. (mg/L)

Frequency of callus (%) Weight of callus (g)

Type of callus

V1 V2 V3 V1 V2 V3 V1 V2 V3

MSC1 MSC2 MSC3

2,4-D

2.0 3.0 4.0

50% 90% 75%

60% 95% 80%

45% 80% 70%

1.0±0.24e 2.6±0.63a 2.0±0.51bc

1.0±0.30e 2.64±0.65a

2±0.51c

0.7±0.06e 2.4±0.75a 1.0±0.24

Smooth compact Nodular compact Smooth compact

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth nodular Smooth compact

MSC4 MSC5 MSC6

2,4-D+BAP

3.0+1.0 3.0+1.5 3.0+2.0

70% 80% 75%

80% 85% 70%

70% 75% 65%

2±0.51d 2.5±0.75ab 2.2±0.63b

2.0±0.51c 2.54±0.78ab 2.3±0.69b

1.5±0.50cd 2.3±0.69ab 1.2±0.30c

Smooth compact Nodular compact Smooth compact

Smooth compact Nodular compact Smooth compact

Smooth compact Smooth friable Smooth compact

MSC7 MSC8 MSC9

2,4-D+Kin

5.0+2.0 5.0+2.5 5.0+3.0

70% 70% 70%

70% 75% 70%

60% 65% 60%

2.0±0.45c 2.1±0.57cd 1.2±0.31de

2.0±0.56cd 2.2±0.57bc 1.2±0.31d

2.0±0.57bc 2.0±0.51b 1.0±0.24d

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth friable Smooth compact

LSD 1.65 1.95 2.20

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Table 3. Influence of growth hormones on callus induction from nodal explants.

Each value is mean of twenty replicates with standard error (mean ± S.E). Mean along with different letters in the same column vary significantly at P =0.05 according to Duncan’s new multiple range test.

Table 4. Effect of different media on shoot initiation from nodal explants in Grapes.

Each value is mean of twenty replicates with standard error (mean ± S.E) a, b, c. Mean along with different letters in the same column vary significantly at P =0.05 according to Duncan’s new multiple range test.

Medium

Growth regulator

Conc. mg/L

Frequency of callus (%) Weight of callus (g) Type of callus

V1 V2 V3 V1 V2 V3 V1 V2 V3

MSC10 MSC11 MSC12

BAP+2,4-D

1.0+2.0 1.0+3.0 1.0+4.0

65% 85% 40%

70% 90% 45%

60% 80% 40%

2.0±0.51d 3.0±1.04b 2.4±0.68bc

2.1±0.53de 3.2±1.10b 2.5±0.75c

1.9±0.47d 2.8±0.92b 2.3±0.63c

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth friable Smooth compact

MSC13 MSC14 MSC15

NAA+BAP

1.0+0.5 1.0+0.25 2.0+0.25

80% 90% 70%

80% 95% 70%

80% 80% 70%

2.1±0.53cd 3.4±1.32a 2.3±0.63bc

2.2±0.57d 3.6±1.38a 2.4±0.68cd

2.0±0.51cd 3.2±1.12a 2.2±0.57c

Smooth compact Nodular compact Smooth compact

Smooth compact Nodular compact Smooth compact

Smooth compact Nodular compact Smooth compact

MSC16 MSC17 MSC18

NAA

0.25 0.5 1

60% 70% 50%

60% 75% 55%

60% 65% 50%

1.9±0.47de 2.2±0.57c 1.2±0.21e

2.0±0.51de 2.3±0.63d 1.2±0.21e

1.8±0.41de 2.1±0.53cd 1.2±0.21e

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth friable Smooth compact

Smooth compact Smooth friable Smooth compact

LSD 2.27 2.38 2.16

Medium Growth regulator

Conc. (mg/L)

No. of days taken for shoot initiation Shoot initiation (%) Mean length of shoots (cm)

V1 V2 V3 V1 V2 V3 V1 V2 V3 MS1 MS2 MS3

BAP

0.25 0.5 1.0

30±16.63ab 22±12.01d

18.6±7.122e

21±10.88cd 18±7.107e 15±7.98e

30±16.634b 25±13.74cd 19±7.113e

45% 45% 85%

45% 50% 90%

30% 40% 80%

2.2±0.57de 3.0±1.04c 4.2±1.73a

2.2±0.57e 3.1±1.09c 4.5±1.79a

2.0±0.51e 2.8±0.92c 4.0±1.63a

MS4 MS5 MS6

BAP+NAA 3.0+0.5 2.0+0.5 1.0+0.5

29±16.05b 25±13.74c 24±12.01cd

25±13.749ab 22±12.01c 20±10.86d

33±17.84ab 28±16.057c 24±13.43cd

50% 60% 75%

50% 60% 75%

50% 60% 75%

1.9±0.40e 2.8±0.92d 3.1±1.50c

2.7±0.71de 2.9±0.98cd 2.9±1.56cd

2.2±0.57e 2.5±0.75d 2.6±1.38d

MS7 MS8 MS9

BAP+TDZ 2.5+1.0 2.5+2.5 2.5+3.0

32±17.78a 23±10.98cd 18±7.107e

30±16.63a 20±10.86d 17±7.101e

35±17.93a 24±10.98d 20±10.86e

60% 70% 75%

60% 70% 75%

60% 70% 75%

2.9±0.98d 3.0±1.04c 3.5±1.32b

2.9±0.98cd 3.2±1.10c 3.8±1.50b

2.4±0.68de 2.9±0.98c 3.5±1.32b

LSD 24.7 20.01 20.8 3.03 2.87 2.92