Effect of media type and explants source on ... - Academic Journals

Journal of Medicinal Plants Research Vol. 6(9), pp. 1742-1751, 9 March, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1551 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Effect of media type and explants source on micropropagation of Dalbergia sissoo: A tree of medicinal importance Aamir Ali*, Muhammad Rizwan, Abdul Majid, Asif Saleem and Naima Huma Naveed Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan. Accepted 14 December, 2011

Attempts were made to evaluate suitable cultural conditions for micropropagation of Dalbergia sissoo from nodal meristem. The best shoot formation response (88%) was obtained on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 1.0 mg/l 6-benzylamino-purine (BAP) + 0.25 mg/l α- naphthalene acetic acid (NAA) after 10.8 days of explants inoculation and average shoot length was recorded to be 2.4 cm after 25 days . For in vitro shoot multiplication, MS medium inferred with 1.5 mg/l BAP + 0.25 mg/l Kinetin exhibited maximum number of shoot, that is, 4.0 shoots per culture vial were formed with an average shoot length of 1.78 cm after 4 weeks of inoculation. For rooting of well developed in vitro shoots, optimum results were obtained on MS medium supplemented with 1.0 mg/l of indole butyric acid (IBA) which were 3.4 roots per plant with an average root length of 1.8 cm after 24 days of inoculation. During all this investigation, different temperature conditions ranging from 23 to 30°C were provided to the cultures, however, optimum results were obtained at 26 ± 1°C at 16/8 h (light/dark) photoperiod. Key words: Dalbergia sissoo, tissue culture, micro propagation, culture media, shoot multiplication, in vitro rooting.

INTRODUCTION Shisham (Dalbergia sissoo) is one of the most important woody plants. It is also called sissoo, sisu, sheesham, tahli. It is primarily found growing along river banks at 900 m elevation, but can range naturally up to 1500 m (Joshi et al., 2003). D. sissoo, not only provide shade and shelter but is also utilized as timber and fuel wood (Shah et al., 2010). Its heart wood is resistant to termites. Soft wood of D. sissoo is used for plywood. Charcoal is also made from it which is used in heating and cooking. Fibers, alkaloids, tannins and resins are also obtained from it. D. sissoo is not only a timber tree, but it also has

*Corresponding author. E-mail: [email protected]. Abbreviations: MS, Murashige and skoog’s medium; BAP, 6benzylamino-purine; Kin, kinetin; IAA, indole acetic acid NAA, α- naphthalene acetic acid, IBA, indole butyric acid; HCl, hydrochloric acid; NaOH, sodium hydroxide.

many medicinal properties. It has been used as aphrodisiac, abortifacient, antipyretic, anthelmintic and expectorant (Hajare et al., 2000). It is used to treat ulcers, skin diseases, stomach troubles, dysentery, emesis and leucoderma (Sharma et al., 2001; Ishtiaq et al., 2006; Ahmad, 2007). D. sissoo is antidiahorrhoeal as it affects bacterial virulence. However, it has no microbial activity (Brijesh et al., 2006). Roots, bark and leaves are used as stimulant and astringent (Hussain et al., 2008). Boiled leaf filtrate of this plant is also used to wash hair for removing dandruff and also to make hair grows (Sultana et al., 2006). D. sissoo is generally propagated through seed which is not a reliable method of propagation because of poor germination and death of young seedlings under natural environmental conditions (Chaudhary and Hameed, 2003). Plants propagated through seed also show variation. To better improve the competitive capacity for producers, new high value commodities of forest trees are needed to be developed in order to supply the domestic as well as foreign demand

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and also to balance the import force. Conventional breeding methods are too difficult and time consuming. Therefore, there is a dire need to introduce some advanced methods not only for mass scale propagation but also for varietal improvement. Among advanced methods of plant propagation, in vitro culture or micropropagation has been established as a method for mass scale propagation of plants and really promising for the production of good quality seedlings in relatively shorter span of time. This technique also provides increased genetic variability in relatively shorter time without applying a sophisticated technology. Now a days this technique is being used for the improvement of many forest trees (Gyvess et al., 2007; Rathore et al., 2007) especially by somaclonal variation, in vitro induced mutagenesis and genetic transformation through callus culture. The aim of this study was to standardize in vitro micropropagation system for D. sissoo by modifying the composition of plant growth regulators (Auxins and cytokinins) in the basal medium during the different stages of the culture. Main criteria used to select best micropropagation conditions were improvement of multiplication rates, shoot quality and rooting efficiency.

MATERIALS AND METHODS The study was undertaken at the University of Sargodha, Sargodha. Explants were taken from Shisham (D. sissoo) plants present in main campus. All kinds of shoots that is, (i) Young (ii) soft and comparatively mature and hard were collected. Nodal segments measuring 1.5 to 2.0 cm were cut and thoroughly washed with running tap water for 5 to 6 min to remove all dust particles. Explants were then treated with household detergent for 10 min. This was followed by second washing with tap water to remove all traces of detergent. After this, explants were dried with the help of sterilized tissue papers. Sometimes, explants were treated with 0.05% Tweene-20 solution for 10 min to break surface tension. Again washed 3 to 4 times with sterilized distilled water to remove all the traces of Tweene-20. Explants were then treated with a solution containing 2% sucrose + 1.5% ascorbic acid + 1.5% citric acid for 10 min to avoid the exudation of phenolic compounds in growth medium, washed three times with sterilized distilled water to remove all the traces of sucrose. After this step explants were treated with 1% solution of Sodium hypochlorite for 15 min. Finally, it was washed 3 times with sterilized distilled water to remove all traces of sodium hypochlorite. Murashige and Skoog (1962) basal medium was used throughout this study. For shoot formation and multiplication MS media was supplemented with growth regulators such as BAP, Kinetin (6furfuryl amino purine) and NAA either alone or in combination with each other in different concentrations. For rooting, MS basal medium was used with different concentrations of auxins that is, NAA, IBA and IAA. The medium was solidified with 0.7 % agar and pH of medium was adjusted between 5.7 to 5.8 with 0.1 N solution of NaOH or HCl. Media was autoclaved at 121°C and 15 Ibs/inch2 pressure for 15 to 20 min. Cultures were incubated in a culture room in which temperature ranging from 26±1˚C to 30±1˚C was tested and optimim temperature was maintained with 16 h light and 8 h dark period in every 24 h cycle. Cultures were maintained under fluorescent light having 2500 lux intensity. The data was recorded for days to shoot formation, shoot length, number of shoots per

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culture vial, days for root induction, length of root and number of test tubes showing response. The experimental design was completely randomized with five replicate cultures for each hormonal treatment and each experiment was repeated thrice. Duncan’s New Multiple Range tests for significance between mean were applied for statistical analysis (Steel and Torrie, 1980). Data was also subjected to Analysis of Variance (ANOVA). Data were analyzed using CoStat statistical software (CoHort Software, Berkeley, CA).

RESULTS AND DISCUSSION The main aim of the present study was to standardize protocols for in vitro propagation of Shisham (D. sissoo) to ensure the easy availability of identical, disease free and premium quality planting material. In the present study, nodal segments from different positions of shoot of different age were taken from well developed field grown plant to find out most appropriate portion to be used as explants. It was observed that nodal meristem with soft and green buds (2 weeks old shoots) showed rapid and high rate of shoot formation response (88%) as compared to explants taken from mature parts (6 month or older shoots) of the plant with brownish buds (Table 1). This may be due to exudation of more phenolic compounds from mature parts of the plant. These results reflects that explants juvenility is an essential parameter for successful in vitro culture of woody plants. Thirunavoukkarasu et al. (2010) while working on D. sissoo reported similar findings which further strengthen our findings. As for as optimum temperature for in vitro propagation is concerned, different scientists have reported different temperature conditions, Joshi et al. (2003) reported that optimum results were obtained at 23±2°C while Thirunavoukkarasu et al. (2010) reported 25±2°C as best temperature for in vitro propagation of D. sissoo. However, present investigation, 26±1°C was found to be the most appropriate temperature for in vitro shoot formation and growth. This variation in temperature may be due to variation in genotype or type of explants used (Ali, 2008).

In vitro shoot formation MS medium was used either in its basal form or in combination with different cytokinins and auxins for efficient shoot formation from nodal meristem. Data presented in Table 2 shows that when MS basal medium was used only 24% shoot formation response was obtained after 23.8 days of nodal meristem inoculation. Growth and morphogenic responses of in vitro cultures depend among other factors, on the correct constituents and balances of growth regulators (Lakshmi et al., 2006). Therefore in present investigation, different concentrations and combinations of auxins and cytokinins were used in MS medium to find out their role for in vitro

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Table 1. Effect of light and dark regimes on in vitro shoot formation.

Photoperiod (light/dark hour)

No of test tubes cultured

16/8 18/6 20/4 8/16 10/14 12/12 LSD (0.05)

10 10 10 10 10 10

No of test tubes showing shoot formation Explants with soft, Explants with green buds mature buds a a 8.8±0.48 7.6±0.4 b b 7.6±0.48 6.6±0.8 c c 6.4±0.8 5.6±0.48 d d 5.0±0.63 4.4±0.8 c c 6.2±0.4 5.2±0.63 bc bc 6.8±0.4 5.4±0.48 0.808 1.092

Days for shoot formation Explants with soft, Explants with green buds mature buds a a 10.8±0.74 12.4±0.4 b b 12.4±1.49 14±0.89 b b 13.8±1.6 14.8±0.6 d d 21±0.89 22.4±0.8 c c 16.5±0.44 18.2±0.4 c c 15.4±0.48 17.2±0.48 1.53 1.43

Rate of Shoot formation (%) Explants with soft, Explants with green buds mature buds 88 76 76 66 64 56 50 44 62 52 68 54

Means followed by different letters in the same column differ significantly at P=0.05 according to Duncan’s new multiple range tests.

shoot formation. Ammirato (2004) reported that cytokinin at moderate concentrations enhance shoot development. Among two cytokinins used, the response of BAP was found to be better than Kinetin that is, 88% shoot formation was obtained after 14.8 days of explants inoculation when 2.0 mg/l BAP was used in MS medium. The average shoot length was found to be 2.04 cm after 25 days of explants inoculation (Table 2 and Figure 1). By increase or decrease in the concentration of BAP the rate of shoot formation was decreased. In case of Kinein, MS medium containing 1.0 mg/l of Kin, provided 84% shoot formation response after 9.6 days of explants inoculation with an average shoot length of 1.7 cm after 25 days of inoculation. Thirunavoukkarasu et al. (2010) obtained efficient (77%) shoot formation response on MS medium containing 6.6 μM BA. Better response of BAP over Kinetin during in vitro propagation of D. sissoo has also been reported by Behera et al. (2008) and Nayak et al. (2007). Similarly Kavyashree (2007) also found same results during in vitro shoot formation of Mullbery.

As for as the effect of different combinations of cytokinins (BAP + Kinetin) in MS medium is concerned, MS medium supplemented with 1.5 mg/l BAP + 0.25 mg/l Kinetin resulted in 80% shoot formation response after 12.1 days of explants inoculation with an average shoot length of 1.4 cm. Auxin/cytokinin ratio during in vitro tissue culture can play a critical role to induce the morphogenic response in higher plants (Garcia et al., 2008). Result of present study also highlights the effect of cytokinins + auxins on in vitro shoot formation. Data presented in Table 2 shows that when MS medium containing 1.0 mg/l BAP + 0.25 mg/l NAA was used, 88% shoot formation response was obtained after 10.8 days of inoculation with an average shoot length of 2.4 cm. Joshi et al. (2003) also reported best that is, 75% shoot formation response in same combination (1.0 mg/l BAP + 0.25 mg/l NAA) in D. sissoo. Thirunavoukkarasu et al. (2010) obtained optimum shoot formation response (76.9%) from axillary buds derived from coppice shoots in MS medium inferred with 6.6

μM BA + 0.57 μM IAA. From data presented in Table 2 it becomes obvious that overall best medium for in vitro shoot formation was MS medium inferred with 1.0 mg/l of BAP + 0.25 mg/l of NAA that is, 88% shoot formation response was obtained after 10.8 days of explants inoculation with an average shoot length of 2.4 cm (Table 2 and Figures 1, 2 and 3). Same rate of shoot formation response (88%) was also obtained in MS medium containing 2.0 mg/l BAP but time taken in this medium was higher that is, 14.8 days after inoculation.

In vitro shoot multiplication For multiplication of in vitro developed shoots MS medium was supplemented with different concentrations of BAP, Kinetin and NAA either alone or in combination with each other. Data presented in Table 3 shows that when 2 mg/l BAP was used in MS medium, an average of 3.2 shoots per culture vial were formed after four

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Table 2. Effect of different hormones on in vitro shoot formation.

S/N

Media (MS)

SB1

MS medium

SB2 SB3 SB4 SB5 SB6 SB7 SB8 SB9 SB10 SB11 SB12 SB13 SB14 SB15 SB16 SB17 SB18 SB19 SB20

MS+BAP

MS+Kin

MS+BAP+NAA

MS+BAP+Kin

Concentration (mg/l)

No of test tubes Cultured

Days for shoot formation

5

23.8±1.16

0.5 1.0 1.5 2.0 2.5

5 5 5 5 5

21±1.26 gh 18.6±0.8 g 16.6±0.8 c 14.8±0.97 g 17.8±1.16

0.5 1.0 1.5 2.0

5 5 5 5

10.8±0.74 a 9.6±0.48 cd 11±0.89 ef 12±0.63

b

0.50+0.25 1.0+0.25 1.5+0.25 1.5+0.5 2.0+0.25

5 5 5 5 5

14.8±0.74 f 10.8±0.4 de 11.4±0.8 f 15±0.89 g 16±1.09

f

1.0+0.25 1.0+0.50 1.5+0.25 1.5+0.50 2.0+0.25

5 5 5 5 5

15±0.89 f 14.8±0.74 ef 12.1±0.2 f 15.2±0.74 g 16±1.09 1.222

LSD (0.05)

h

gh

f

*Shoot length (cm)

No of test tubes showing shoot formation

i

1.2±0.4

1.04±0.48 efgh 1.16±0.14 c 1.45 ±0.1 a 2.04 ±0.25 cdef 1.3±0.12

hi

1.32±0.17 b 1.7±0.10 efgh 1.22±0.04 gh 1.12±0.74

cde

3.4±0.48 a 4.2±0.48 cde 2.6±0.48 def 2.2±0.4

1.15±0.08 cd 2.4±0.10 ghi 1.06±0.08 ghi 1.1±0.06 ghi 1.1±0.08

efgh

3±0.63 a 4.4±0.8 cde 2.6±0.48 def 2.2±0.74 ef 2±0.63

fgh

2.9±0.2 bc 3.4±0.48 ab 4.0±0.63 cd 3.0±0.31 def 2.2±0.4 0.721

0.94±0.08

1.13±0.10 defg 1.23±0.04 cd 1.4±0.00 fgh 1.14±0.04 ghi 1.1±0.08 0.155

Rate of shoot formation (%)

g

24

1.6±0.48 def 2.4±0.48 cde 2.8±0.4 ab 4.4±0.4 bc 3.4±0.48

fg

32 48 56 88 68

bc

68 84 52 44

cd

60 88 52 44 40

cd

58 68 80 60 44

Means followed by different letters in the same column differ significantly at P=0.05 according to Duncan’s new multiple range tests.* Shoot length recorded after 25 days of explants inoculation.

weeks of inoculation with an average length of 1.98 cm. Table 3 further depicts that by increase or decrease in the concentration of BAP beyond 2 mg/l, the rate of shoot multiplication was gradually

decreased. When Kinetin was used in MS medium no good multiplication response was obtained. Using high concentrations of cytokinins could produce hyperhydric shoots with a low

ability to growth (Castro-Concha et al., 1990). This could support the idea of reducing BAP levels to enhance cytokinin: auxin synergisms instead of cytokinin shocks, as a way to improve plant

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Figure 1. Shoot initiation from nodal explants after 11 days of inoculation on MS medium containing 1.0 mg/l of BAP + 0.25 mg/l of NAA (1.5×) (1×).

Figure 2. In vitro Shoot growth after 30 days of inolation on MS medium containing 1.0 mg/l BAP + 0.25 mg/l NAA (5×).

Figure 3. Shoot initiation from multi node explants after 25 days of inoculation on MS medium containing 1.0 mg/l BAP + 0.25 mg/l NAA (1×).

regeneration. To study the effect of cytokinin-auxin synergism for in vitro shoot multiplication, BAP was tested with NAA in MS medium, it was noticed that in

vitro shoot multiplication response was comparatively better as compared to BAP alone (Figures 4 and 5). A combination of 1.0 mg/l BAP + 0.25 mg/l NAA resulted

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Table 3. Effect of different hormones on in vitro shoot multiplication.

No of test tubes cultured

*No of shoots per culture vial

5

1.1±0.2

MS+ BAP

1.0 1.5 2.0 2.5 3.0

5 5 5 5 5

1.6±0.48 de 2.6±0.48 bcd 3.2±0.74 efg 2.2±0.74 fgh 1.8±0.4

MS+Kin

1.0 1.5 2.0 2.5

5 5 5 5

MS+BAP+NAA

0.5+0.25 1.0+0.25 1.0+0.5 1.5+0.25 1.5+0.5

5 5 5 5 5

2.6±0.48 ab 3.8±0.40 bcd 3.2±0.4 cd 3±0.63 def 2.4±0.48

1.0+0.25 1.0+0.50 1.5+0.25 1.5+0.50 1.0+1.0

5 5 5 5 5

2.9±0.2 abc 3.4±0.48 a 4.0±0.0 bcd 3.1±0.2 def 2.4±0.48 0.681

S/N

Media (MS)

M1

MS medium

M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15

M16 M17 M18 MS+BAP+Kin M19 M20 LSD (0.05)


h

*Average shoot length (cm) 1.02±0.04

j

gh

1.32±0.07 def 1.42±0.19 a 1.98±0.29 b 1.76±0.16 cd 1.56±0.10

2.8±0.4 efg 2.2±0.4 fgh 1.8±0.74 h 1.4±0.48

cde

1.38±0.07 fgh 1.28±0.04 hij 1.16±0.04 ij 1.08±0.04

de

1.22±0.04 bc 1.68±0.14 efgh 1.34±0.08 efgh 1.34±0.10 defg 1.4±0.14

cde

efgh

defg

ghi

efgh

1.34±0.10 de 1.5±0.00 b 1.78±0.04 defg 1.4±0.14 fgh 1.3±0.00 0.165


Figure 4. Shoot growth and development from multi- node explants after 45 days of inoculation on MS medium containing1.0 mg/l BAP+0.25 mg/l NAA.

3.8 shoots per culture vial with an average shoot length of 1.68 cm after four weeks of inoculation on this medium. Any increase or decrease in the concentration

of either BAP or NAA beyond this level not only decreased shoot multiplication response but average shoot length was also less. Our results are in agreement

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Figure 5. Initiation of shoot multiplication (buds) on MS medium containing 1.5 mg/l BAP+0.25 mg/l Kinetin after 50 days of inoculation (1.5×)

Figure 6. Multiple shoot formation on MS medium after 60 days of inoculation on MS medium containing 1.5 mg/l BAP + 0.25 mg/l Kinetin (1.5×).

with previous in vitro studies of Joshi et al. (2003) on D. sissoo who reported maximum 3.67 multiple shoots per culture vial with same concentrations and combination of BAP and NAA in MS medium. Bhojwani (2008) studied the in vitro shoot multiplication of hybrid willow tree and also reported that when the concentration of BAP was decreased beyond a certain level, the rate of shoot multiplication increased. Growth and morphogenesis in vitro is regulated by interaction and balance between the growth regulators supplied in the medium and the growth substances produced endogenously by the cultured cells. Apart from the direct effect on cellular mechanisms, many synthetic growth regulators may intact modify the level of endogenous substances (George and Sherrington, 1984). As for as the synergy between BAP and Kinetin in present study is concerned, MS medium containing 1.5 mg/l BAP + 0.25 mg/l Kinetin proved best for in vitro shoot multiplication. In this combination average 4.0 shoots per culture vial with an average shoot length of 1.78 cm were formed (Table 3 and Figures 6 and 7). These conclusions are against the previous reported

findings of Joshi et al. (2003), who obtained poor shoot multiplication response in BAP + Kinetin combination. According to them maximum number of shoots (8.04 shoots per culture vial) were formed on MS medium containing 1.0 mg/l BAP+0.25 mg/l NAA after 150 days of meristem inoculation while Pradhan et al. (1998) reported maximum number of shoots per culture vial and highfrequency shoot proliferation (99%) with 8.9 μM BAP. This difference in results may be due to difference in genotype each having different hormonal requirements. From data presented in Table 3, it is clear that MS medium containing 1.5 m/g/l BAP + 0.25 mg/l Kinetin showed best shoot multiplication response which was 4.0 shoots per culture vial with an average shoot length of 1.78 cm (Figures 7 and 8).

In vitro rooting In the present study although roots were formed on MS basal medium, but number of roots per plants were less

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Figure 7. In vitro shoot multiplication after 42 days of inoculation on MS medium containing 1.5 mg/l BAP + with 0.25 mg/l Kin (1×).

Figure 8. Well developed multiple shoots formed after 50 days of inoculation on MS medium containing 1.5 mg/l BAP + 0.25 mg/l Kinetin (2×).

and time taken for root formation was also more that is, only 1.4 roots per plant were formed with an average root length of 0.9 cm after 28.6 days of inoculation in rooting medium MS basal medium but number of roots per plants were less and time taken for root formation was also more that is, only 1.4 roots per plant were formed with an average root length of 0.9 cm after 28.6 days of inoculation in rooting medium. In present study although roots were formed on MS basal medium but number of roots per plants were less and time taken for root formation was also more that is,. only 1.4 roots per plant were formed with an average root length of 0.9 cm after 28.6 days of inoculation. In some earlier reports, varying concentrations of different auxins have been used for in vitro root induction (Joshi et al., 2003; Chand and Singh, 2005; Hegazi and Gabr, 2010) from micro shoots of D. sissoo. In present study too, supplementation of varying concentrations of different auxins (IAA, IBA and NAA) in MS medium was found to be an essential factor for rooting of in vitro developed shoots. When MS medium

was supplemented with different concentrations of IAA, it was noticed that at 1.0 mg/l IAA, 3 roots per plant were formed after 24.4 days of inoculation with an average root length of 1.6 cm. Further increase or decrease in the concentration of IAA not only produced less roots but time taken for rooting was also more. When NAA was used in MS medium, results were almost similar as that of IAA. Best results for in vitro rooting were obtained on MS medium inferred with 1.0 mg/l of IBA that is, 3.4 roots per plants were formed after 24 days of inoculation with an average root length of 1.8 cm (Table 4). These results are in agreement with the results reported by Joshi et al. (2003). who used three different auxins (IBA, NAA, and IAA) individually in varying concentrations for in vitro rooting of D. sissoo and reported best rooting response (76.92%) at 1.0 mg/l IBA followed by 69.23and 61.84% in NAA and IAA, respectively. They further reported that the concentration of IBA higher than 1.0 mg/l decreased the in vitro rooting response. Similarly, Thirunavoukkarasu

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Table 4. Effect of different concentrations of IAA, IBA and NAA on in vitro root formation.


No of test tubes Cultured

Day for root formation g

MS+IAA

0.5 1.0 1.5 2.0

5 5 5 5 5

28.6±0.48 bcd 26±1.09 abc 24.4±1.2 cde 26.4±0.8 fg 27.4±0.48

1.4±0.48 bcd 2.5±0.44 ab 3.0±0.63 bcd 2.4±0.48 cde 2.0±0.63

5 5 5 5

cd

26.2±0.74 ab 24±0.63 def 26.6±1.01 fg 27.8±1.16

bcde

MS+IBA

0.5 1.0 1.5 2.0

2.2±0.4 a 3.4±0.44 bcd 2.4±0.48 de 1.8±0.74

1.32±0.14 a 1.8±0.25 bcde 1.36±0.10 gh 1.02±0.04

0.5 1.0 1.5 2.0

5 5 5 5

25.4±1.01 bcd 24.6±1.01 abc 24.4±1.16 efg 26.6±1.2 1.363

bcd

2.4±0.48 bcd 2.5±0.44 abc 2.8±0.4 bcde 2.2±0.4 0.729

bcd

1.4±0.32 bc 1.52±0.20 defg 1.18±0.11 efgh 1.14±0.14 0.233

Code

Media

RF1 RF2 RF3 RF4 RF5

Ms Basal

RF6 RF7 RF8 RF9

RF10 RF11 MS+NAA RF12 RF13 LSD (0.05)

Average number of roots per plant e

Average root length (cm) h

0.9±0.06 fgh 1.1±0.06 ab 1.6±0.17 gh 1.02±0.09 gh 1±0.14 cdef

bcd


et al. (2010) while working on D. sissoo used two auxins (IAA and IBA) and found better results with IBA as compared to IAA that is, maximum 1.3 roots per plant with an average length of 2.40 cm were recorded in the ½ strength MS medium supplemented with 7.35 μM IBA. It was also observed in the present study that by increase in the concentration of auxins rate of root formation was decreased depicting that higher concentration of auxins inhibited rooting. Similar observations were made by Pierik (1987) who opined that higher concentration of auxin inhibits the adventitious and axillary bud formation. It is concluded that best medium for rooting is MS medium inferred with 1.0 mg/l IBA. In this medium 3.4 roots were formed per culture vial having length of 1.8 cm after 24 days of inoculation. REFERENCES Ahmad SA (2007). Medicinal wild plants from Lahore Islamabad motorway (M-2). Pak. J. Bot., 39(2): 355-375 Ali A, Naz S, Siddiqui FA, Iqbal J (2008). An efficient protocol for large scale production of sugarcane through micropropagation. Pak. J. Bot., 40(1): 139-149, 2008. Ammirato PV (2004). Yams In: Ammirato PV, Evans DA, Sharp WR, Yamada Y (eds) Handbook of plant cell culture. Macmillan NY, 3: 329-354. Behera PR, Nayak P, Thirunavoukkarasu M, Sahoo SB (2008). Plant regeneration of Gmelina arborea Roxb. From cotyledonary node explants. Ind. J. Plant Physiol., 13(3): 258-265 Bhojwani SS (1980). Micropropagation method for a hybrid willow (Salix matsudana × alba NZ 1002). N.Z. J. Bot., 18: 209-214. Brijesh S, Daswani PG, Tetali P, Antia NH, Birdi TJ (2006). Studies on Dalbergia sissoo (Roxb) leaves: Possible mechanism(s) of action in infectious diarrhea. Ind. J. Pharmacol., 38(2): 46-48

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