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. 2013 Dec 29;20(1):81–87. doi: 10.1007/s12298-013-0213-3

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

J B Vibha 1, N S Shekhawat 1,✉, Pooja Mehandru 1, Rachana Dinesh 1
PMCID: PMC3925479  PMID: 24554841

Abstract

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO3)2, K2SO4, KCl, and NH4(SO4)2. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.

Keywords: Ex vitro rooting, Regeneration, Dalbergia sissoo, Plant growth regulators, Liquid culture

Introduction

Dalbergia sissoo (shisham) is a multipurpose tree and one of the seven most valuable timber species of India, thus making it an important tree legume for large-scale industrial plantation (Bakshi and Sharma 2011). The bark and wood of D. sissoo are used as an expectorant, antihelminthic, and antipyretic; roots are used as astringent, and leaves are used for eye infection and gonorrhea (Kritikar and Basu 1975). It enriches the soils through nitrogen fixation and with fast decomposing leaves (Bakshi and Sharma 2011). This species also plays an important role in environmental conservation and ecosystem balance (Bari et al. 2008). Therefore, there is a need for rapid propagation of desired genotypes of this forest tree selected for yield and quality of timber.

D. sissoo is conventionally propagated through seeds/planting sucker/stem cutting (Bari et al. 2008). This tree species is open-pollinated and the seed raised plants show wide genetic variability and are genetically not similar to mother plant. This method of propagation does not carry the optimum genetic gain of cloning of selected/mature tree (Bonga and Von Aderkas 1992). Methods of vegetative propagation are laborious, time consuming, and constrained by low multiplication rate. Propagation of woody trees through tissue culture has many advantages over conventional vegetative propagation method like fast multiplication of the elite genotypes, quick release of improved cultivars, production of disease-free plants, season-independent production of plants, germplasm conservation, and facilitating their easy exchange (Pena and Seguin 2001; Asthana et al. 2011). In vitro approach to clone selected and mature trees(s) of D. sissoo can be applied as an efficient tool for micropropagation.

Earlier, micropropagation of D. sissoo using seedling-derived explants has been reported by a number of workers (Das et al. 1997; Pattnaik et al. 2000; Singh et al. 2002; Singh and Chand 2003; Chand and Singh 2004, 2005; Bari et al. 2008). The disadvantage of using juvenile rather than adult/selected specimen for propagation is that the full genetic developmental potential of the former is less known than that of adult/mature plant (Bonga and Von Aderkas 1992; Pijut et al. 2012). Till date, in vitro plant regeneration of D. sissoo from explants of mature tree has also been reported, but these explants produced callus (Datta and Datta 1983) and less number of shoots (Joshi et al. 2003; Thirunavoukkarasu et al. 2010). Datta et al. (1982) and Gulati and Jaiwal (1996) rooted the microshoots of D. sissoo under in vitro condition by two-step method. Generally, woody plant species are recalcitrant to adventitious regeneration during their maturation stage as the vigor for shoot production and competence for rooting declines (Singh et al. 2002; Bonga et al. 2010). D. sissoo reaches maturity after 20–25 years in the arid regions. Selection of desired genotype for quality and quantity of timber yield is possible at this age. We report here an improved micropropagation protocol from mature tree of D. sissoo through axillary bud proliferation from nodal shoot segment followed by successful transplantation of ex vitro rooted plantlets. Ex vitro rooting of shoots is advantageous as compared to in vitro rooting of plantlets as it results in better root system, requires less time, and allows acclimatization with ease. The cost of tissue culture raised plants can be reduced by such amendment in production process (Yan et al. 2010; Phulwaria et al. 2012a).

Materials and methods

Explants preparation and surface sterilization

About 20–25-year-old tree of D. sissoo was selected for establishment of cultures. The selected tree was lopped during winter (December–January). The flushed/rejuvenated shoots produced during subsequent spring (March–April) were harvested and used as explant. Fresh shoot sprouts (4–5-cm long with 2–4 nodes) were washed thoroughly and pretreated with 0.1 % (w/v) bavisitin (a systemic fungicide; BASF India Ltd., Mumbai, India) for 15 min followed by surface sterilization with 0.1 % (w/v) HgCl2 (Hi-Media, India) for 3–5 min under aseptic conditions. Each treatment was followed by rinsing with sterile water 4–5 times. The nodal shoot explants were treated with chilled sterile antioxidant solution (0.1 % each of ascorbic acid and citric acid) for 15 min.

Nutrient media

Murashige and Skoog (1962) medium with sucrose (3 %) and 0.8 % (w/v) agar–agar (Bacteriological grade, Qualigens Fine Chemicals, Mumbai, India) was used for culture. The pH of the medium was adjusted to 5.8 ± 0.02 prior to autoclaving for 15 min at 121 °C.

Bud breaking and multiple shoot initiation

Surface-sterilized explants were inoculated vertically on the culture medium with various concentrations (0.0, 4.44, 8.88, 13.32, 17.76 μM) of BAP and (4.65, 9.28, 13.92, 18.56 μM) of Kn. The cultures were incubated in culture room under controlled conditions of temperature (26 ± 2 °C), 12 h d−1 illuminations of PFD 30–40 μ mol m−2 s−1 and 60 % relative humidity (RH).

Multiplication of shoots

The explants with regenerated shoots were repeatedly transferred to fresh nutrient medium with different concentrations (1.11, 2.22, 4.44 μM) of BAP or (1.16, 2.32, 4.65 μM) Kn or in combination of 2.22 μM of BAP with 0.46, 2.32, 4.65 μM of Kn and 0.002, 0.004 μM of TDZ. The effect of different media, namely WP, (Lloyd and McCown 1981), MMS (Shekhawat et al. 1998), and MS (Murashige and Skoog 1962), with different strengths (full, half, and one fourth) on shoot multiplication were also evaluated. Each medium was supplemented with 2.22 μM of BAP, 0.002 μM of TDZ, and 1 mM each of calcium nitrate, ammonium sulfate, potassium chloride, and potassium sulfate.

In another set of experiments, the in vitro regenerated shoots were transferred to half-strength MS liquid medium with the abovementioned salts and Plant growth regulators (PGRs). The shoots were cultured in 10 to 20 ml liquid medium in 150-ml flask or 10, 20, or 30 ml liquid medium/250-ml flask. These were kept on rotatory shaker, maintained at 80 rpm under high light intensity (PFD 45–50 μmol m−2 s−1). The cultures were further multiplied by (1) the transfer of mother explants, after harvesting newly formed shoots, to optimized shoot multiplication medium (liquid MS medium) for four passages and (2) subculturing of excised newly formed shoots on fresh medium. Repeated transfer was done after every 15–17 days.

Ex vitro rooting of microshoots and hardening of plantlets

About 3–4 cm long microshoots were harvested and treated with indole-3-butyric acid (IBA) (0, 492, 984, 1,476 μM), NAA (537, 1074, 1611 μM), NOA (495, 989, 1484 μM), and a combination of IBA (246, 492, 738 μM) with NAA (247, 495, 742 μM) for 2-min. The treated shoots were transferred to autoclaved soilrite (a mixture of horticulture grade perlite with Irish peat moss and exfoliated vermiculite supplied by Kel Perlite, Bangalore, India). Shoots were moistened with one-fourth strength of MS salt solution. The culture bottles were capped and placed in the greenhouse near the pad section (RH 80–90 % and 28 ± 2 °C). After initiation of the root(s), the plantlets were gradually shifted from the pad section towards the fan section (temperature 32 ± 2 °C; 60–70 % RH) and the caps of bottles gradually loosened and finally removed after 15–20 days. Finally, the plants were transferred into polybags containing a mixture of organic manure, garden soil, and sand (1:1:1) and kept under greenhouse conditions at 25–30 °C, 80–90 % RH. Rooting was recorded after 2–3 weeks.

Experimental design and data analysis

The experiments were set up in completely randomized block design and repeated thrice. All the experiments were conducted with a minimum of 20 replicates per treatment. To confirm the reliability of the method, the experiments were repeated with four other local accession of D. sissoo. The number and length of shoots as well as roots were scored after regular intervals. The data was analyzed statistically using SPSS v. 17 (SPSS, Chicago, USA). The significance of differences among means was carried out using Duncan’s multiple range tests at P < 0.05. The results are expressed as means ± SD of three experiments.

Results and discussion

Establishment of cultures

The cultures could be established from the nodal shoots of explants derived from fresh flushed/rejuvenated shoot of spring harvested from mature tree (Fig. 1a) and lopped during the preceding winter. The ability to clone woody plants is greatly influenced by the maturation stage and by the physiological stage of the initial explants (Montenius 1987). In the present study, nodal segments with 1–2 nodes, (4–5 cm in length) derived from rejuvenated (reinvigorated) shoots exhibited bud breaking. The old/perennial shoots of unlopped tree, if cultured, did not respond in vitro. It is suggested that the perennial shoots/tissues experience types of stresses like summer heat, severe winter, and extreme drought during the year, and accumulate anti-metabolic substances like phenols and ABA, which inhibit growth in culture (Shekhawat et al. 1993). Pruning/lopping/pollarding of mature tree and elimination/removal of aged tissues resulted in the production of reinvigorated shoots during subsequent spring. The lopping of tree is important for obtaining explants that respond in culture. Such types of pretreatments to mature trees have also been suggested for reinvigoration and rejuvenation (Bonga and Von Aderkas 1993) of woody plants. Explants harvested from woody plants of the desert area are very sensitive to injuries and sterilization process. Their cut ends show excessive browning and darkening. The treatment of explants with chilled, sterile antioxidant solution checked excessive phenolic exudation and browning of tissue and cultures (Phulwaria et al. 2012a, b). It has been observed in woody plants that antioxidant solutions may promote growth and development of the shoots particularly at the stage of culture establishment (Shekhawat et al. 1993; Sanjaya et al. 2006; Rai et al. 2010). Explants were cultured on 0.8 % agar-gelled MS medium containing BAP or Kn for shoot bud induction. Axillary bud proliferation occurred after 8–10 days of inoculation with 2–3 shoots per node (Fig. 1b). BAP was found to be more effective than Kn in terms of percent response and number of shoots per explants. MS medium supplemented with 8.88 μM BAP was most suitable for multiple shoot induction and it induced 100 % bud break (Table 1). The effectiveness of BAP on multiple shoot induction has also been reported in a number of plants (Rai et al. 2010; Phulwaria et al. 2012a, b).

Fig. 1.

Fig. 1

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In vitro propagation of Dalbergia sissoo. a Mature lopped tree (20–25 year old) of Dalbergia sissoo used as source of explant (scale bar 1.5 m). b Shoot bud induction from nodal explants on MS medium + 8.88 μM of BAP after 8–10 days (scale bar 1 cm). c Multiplication of shoots on MS medium containing 2.22 μM BAP and 0.002 μM TDZ after 20–25 days (scale bar 1 cm). d Shoot elongation and amplification in liquid MS medium with 2.22 μM BAP after 10–15 days (scale bar 1.4 cm). e Ex vitro rooted shoots treated with 984 μM of IBA for 5 min (scale bar 2 cm). f Acclimatized plant of Dalbergia sissoo in greenhouse (scale bar 1.7 cm)

Table 1.

Effect of cytokinins (BAP or Kn) on bud breaking and shoot growth from nodal explants of Dalbergia sissoo

BAP (μM) Kn (μM) Response (%) Shoot no. (mean ± SD) Shoot length (cm) (mean ± SD)
0.0 – 00 0.0 ± 0.0e 0.0 ± 0.0f
4.44 – 50 1.5 ± 0.52cd 1.1 ± 0.06d
8.88 – 100 3.7 ± 0.42a 2.2 ± 0.07a
13.32 – 80 2.8 ± 0.72b 1.5 ± 0.09c
17.36 – 60 1.8 ± 0.63c 0.5 ± 0.17e
– 4.65 0 0.0 ± 0.0e 0.0 ± 0.0f
– 9.28 20 1.2 ± 0.42d 0.6 ± 0.08c
– 13.92 40 1.6 ± 0.51cd 1.6 ± 0.08b
– 18.56 30 1.5 ± 0.53cd 1.4 ± 0.07c
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Means followed by the same letter within columns are not significantly different (P < 0.05) using Duncan’s multiple range test. Data were recorded after 15 days of culture

Multiplication of shoots

Effect of plant growth regulators

The shoots regenerated from the nodal explant were repeatedly transferred to the fresh medium with lower concentration of cytokinin alone (BAP or Kn) or a combination of BAP with Kn and TDZ for shoot multiplication (Table 2). The number of shoots increased on every transfer, reaching the maximum on fourth passage. Tripathi and Kumari (2010) also suggested that the repeated transfer of mother explants suppresses apical dominance allowing induction of basal meristematic cells to form multiple shoots. MS medium supplemented with the combination of 2.22 μM BAP and 0.002 μM TDZ was found best for the multiplication of shoots. A maximum number of 20.9 ± 0.72 shoots (3.9 ± 0.14 cm shoot length) were produced within 15–20 days (Fig. 1c) on this combination. TDZ is among the most active cytokinin-like substances and usually induces greater in vitro shoot proliferation than many other cytokinins, particularly in woody plant species when used in very low concentration (Huetteman and Preece 1993). The amplified cultures of D. sissoo exhibited shoot tip necrosis and defoliation.

Table 2.

Effect of concentrations and combinations of cytokinin (BAP, Kn, and TDZ) on multiplication of shoots of D. sissoo on MS medium

BAP (μM) Kn (μM) TDZ (μM) Shoot no. (mean ± SD) Shoot length (cm) (mean ± SD)
0.0 – – 00 ± 0.0i 00 ± 0.0h
1.11 – – 5.5 ± 0.52f 1.4 ± 0.52f
2.22 – – 12.8 ± 0.72b 2.3 ± 0.76bc
4.44 – – 3.5 ± 0.54h 1.3 ± 0.08d
– 1.16 – 3.6 ± 0.62h 1.8 ± 0.07g
– 2.32 – 3.8 ± 0.72h 1.6 ± 0.60e
– 4.65 – 4.6 ± 0.62g 2.4 ± 0.19b
2.22 0.46 – 7.9 ± 0.73e 1.8 ± 0.92d
2.22 2.32 – 10.5 ± 0.95d 1.5 ± 0.90f
2.22 4.65 – 7.7 ± 0.64e 1.6 ± 0.08e
2.22 – 0.002 20.9 ± 0.72a 3.9 ± 0.14a
2.22 – 0.004 11.9 ± 0.76c 2.3 ± 0.14c
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Means followed by the same letter within columns are not significantly different (P < 0.05) using Duncan’s multiple range test. Data were recorded after 25 days of culture

Effect of different types of culture media

Various culture media with few amendments, i.e., NH4(SO4)2, K2SO4, KCl, and Ca(NO3)2 were evaluated to check the shoot tip necrosis, defoliation, and callus formation. In the present study, half-strength agar-gelled MS medium supplemented with 2.22 μM BAP, 0.002 μM of TDZ, along with 1 mM each of Ca(NO3)2, K2SO4, KCl, and NH4(SO4)2 was the most suitable for multiplication (Table 3). Incorporation of ammonium sulfate in medium reduced hyperhydricity, shoots tip necrosis, and promoted growth of shoots. Effect of ammonium ions and cytokinins on hyperhydricity and rate of multiplication was reported by Ivanova and Staden (2008) in Aloe polyphylla. Sulfate ions promote absorption of nitrate and buffered the culture medium (Ivanova and Staden 2008; Kopriva et al. 2009). Calcium has an important role in cell signaling, acting as a secondary messenger and thus directly influencing the absorption of other nutrients (Peres et al. 2009). The addition of KCl and Ca(NO3)2 help in improving the growth of culture of woody legume. These findings are further supported by earlier reports (Rathore et al. 2004; Husain and Anis 2009). According to Preece (1995), in vitro performance of explants can be improved by balancing the nutrient level in the medium.

Table 3.

Effect of culture media on multiplication of shoots of D. sissoo (each supplemented with BAP (2.22 μM) + TDZ (0.002) and 1 mM each of calcium nitrate, ammonium sulfate, potassium chloride, and potassium sulfate)

Types of media Shoot no. (mean ± SD) Shoot length (cm) (mean ± SD)
MS 6.20 ± 0.91e 2.4 ± 0.20g
MMS 6.90 ± 0.87e 3.0 ± 0.27f
½ MS agar gelled 27.9 ± 0.99b 3.9 ± 0.30c
¼ MS agar gelled 10.4 ± 0.73c 3.5 ± 0.29e
WP 7.90 ± 0.99d 3.5 ± 0.15e
MS liquid 7.20 ± 0.78d 5.2 ± 0.47b
½ MS liquid 29.6 ± 0.70a 6.7 ± 0.46a
¼ MS liquid 6.50 ± 1.50e 3.6 ± 0.27d
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Means followed by the same letter within columns are not significantly different (P < 0.05) using Duncan’s multiple range test. Data were recorded after 25 days of culture

Shoot multiplication in liquid medium

The maintenance of sustainable and high rate of multiplication of healthy shoots was achieved in half-strength liquid medium containing abovementioned salts and PGRs. The shoots multiplied with higher rate (29.6 ± 0.70) and length (6.7 ± 0.46 cm) and without tip necrosis and defoliation (Fig. 1d; Table 3). The rate of shoot amplification achieved for D. sissoo in the liquid medium was higher than reported earlier (Datta and Datta 1983; Gulati and Jaiwal 1996; Joshi et al. 2003; Thirunavoukkarasu et al. 2010). Liquid culture offers many potential advantages over solid cultures like faster growth rates, rapid uptake of nutrients by tissues, and dilution of exuded growth inhibitors, i.e., phenolics released by explants, thus minimizing negative effect on growth (Kim et al. 2003; Gupta and Timmis 2005; Preece 2010). Many factors such as the type, size, and volume of liquid in vessels, time period of repeated transfer, light, and temperature significantly influence the growth of tissue in culture. About 10–15 ml of amended liquid medium in 250-ml flask was found to be optimum for multiplication of cultures. On lesser volume of medium, the growth of cultures was slow and the medium dried up within a week; a volume of medium more than mentioned above caused hyperhydration of shoots, which affected shoot growth and multiplication. Reduction in the number and growth of shoots was due to hyperhydration which is a result of lower oxygen availability and submergence of shoots (Pati et al. 2011). Time period of subculturing was found to be an important factor for the maintenance and multiplication of cultures. Repeated transfer was done after every 15–17 days, as senescence (leaf fall) and browning of media occurred with delay.

Ex vitro rooting and hardening of shoots

In many woody plant species which are difficult to root, the in vitro generated shoots rooted successfully under ex vitro condition and survived (Benmahioul et al. 2012). The in vitro regenerated shoots treated for 2 min with 984 μM of IBA exhibited the highest (90 %) rooting. About 3.7 ± 0.82 number of roots per shoot with 2.8 ± 0.12 cm root length was produced within 3–4 weeks (Fig. 1e; Table 4). IBA has been observed to induce strong rooting response and has been extensively used to promote rooting in a wide range of woody tree species (Eeswara et al. 1998; Husain et al. 2008). Ex vitro rooted plants were successfully acclimatized with 96.6 ± 2.8 % of transplantation survival rate. The ex vitro rooting is not only helpful in reducing resources, time, and labor costs but also simplifies the protocol by eliminating the rooting step under sterile conditions (Preece and Shutter 1991; Dinkel-Meier et al. 1993; Pospisilova et al. 1999; Yan et al. 2010; Phulwaria et al. 2012b). Ex vitro rooting of shoots is advantageous in terms of better root system, time reduction, and ease of acclimatization (Yan et al. 2010; Benmahioul et al. 2012).

Table 4.

Effect of auxins concentrations (pulse treatment) on ex vitro root induction from shoots of D. sissoo

IBA (μM) NAA (μM) NOA (μM) Response (%) Roots no. (mean ± SD) Root length (cm ± SD)
00 – – 0 0 0
492 – – 78 1.5 ± 0.72cd 1.2 ± 0.52f
984 – – 90 3.7 ± 0.82a 2.8 ± 0.12a
1,476 – – 63 1.9 ± 0.56bc 2.6 ± 0.15b
– 537 – 75 1.3 ± 0.48d 1.2 ± 0.10f
– 1,074 – 85 1.1 ± 0.56d 1.8 ± 0.08de
– 1,611 – 79 1.0 ± 0.66d 1.6 ± 0.12e
– – 495 60 1.1 ± 0.61d 0.5 ± 0.09h
– – 989 55 1.1 ± 0.59d 0.8 ± 0.08g
– – 1,484 65 1.0 ± 0.66d 1.2 ± 0.10f
246 – 247 60 1.5 ± 0.73cd 1.8 ± 0.08dc
492 – 495 63 2.0 ± 0.66bc 2.0 ± 0.06d
738 – 742 70 2.2 ± 0.52ab 2.2 ± 0.09c
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Means followed by the same letter within columns are not significantly different (P < 0.05) using Duncan’s multiple range tests. Data were recorded after 25 days

In conclusion, this paper describes a simple and improved protocol for propagation of D. sissoo using liquid medium followed by ex vitro rooting. Following this protocol, about 435 hardened plants could be produced starting with 20 nodal shoot segments. The method described here overcomes constraints like tip necrosis, defoliation, and callus at base by amendment in medium using salts like Ca(NO3)2, K2SO4, KCl, and NH4(SO4)2 followed by the use of liquid medium for elongation of shoots. The use of ex vitro rooting technique for root development in the large-scale commercial production of plantlets seems to be a more appropriate method compared to in vitro rooting. It is an economical method in terms of cost, labor, and time and gives higher yields and profits.

Acknowledgments

Authors Vibha and Rachana acknowledge the support of the University Grants Commission (UGC), New Delhi for the award of Post Doctoral Fellowship for Women. Dr. Pooja Mehandru wishes to thank DST for the award of WOS A fellowship. We also acknowledge the contribution of Dr. Manoj K. Rai (DS Kothari Post Doc Fellow) for his valuable suggestion.

Abbreviations

BAP

6-Benzylaminopurine

IBA

Indole-3-butyric acid

Kn

Kinetin

MS

Murashige and Skoog medium

NAA

Naphthalene acetic acid

NOA

2-Naphthoxy acetic acid

PFD

Photon flux density

PGRs

Plant growth regulators

RH

Relative humidity

TDZ

Thidiazuron

References

  1. Asthana P, Jaiswal VS, Jaiswal U. Micropropagation of Sapindus trifoliatus L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis. Acta Physiol Plant. 2011;33:1821–1829. doi: 10.1007/s11738-011-0721-0. [DOI] [Google Scholar]
  2. Bakshi M, Sharma A. Assessment of genetic diversity in Dalbergia sissoo clones through RAPD profiling. J Forest Res. 2011;22:393–397. doi: 10.1007/s11676-011-0183-7. [DOI] [Google Scholar]
  3. Bari MA, Ferdaus KMKB, Hossain MJ. Callus induction and plantlet regeneration from in vivo nodal and internodal segments and shoot tip of Dalbergia sissoo Roxb. J Bio Sci. 2008;16:41–48. [Google Scholar]
  4. Benmahioul B, Dorion N, Kaid-Harche M, Daguin F. Micropropagation and ex vitro rooting of Pistachio (Pistacia vera L.) Plant Cell Tissue Organ Cult. 2012;41:71–73. [Google Scholar]
  5. Bonga JM, Von Aderkas P. In vitro culture of trees. Dordrecht: Kluwer Academic Publishers; 1992. [Google Scholar]
  6. Bonga JM, Von Aderkas P. Rejuvenation of tissues from mature conifers and its implications for propagation in vitro. In: Ahuja MR, Libby WJ, editors. Clonal Forestry I. Springer-Verlag, Berlin, Germany: Genetics and Biotechnology; 1993. pp. 182–199. [Google Scholar]
  7. Bonga JM, Klimaszewska KK, Von Aderkas P. Recalcitrance in clonal propagation, in particular of conifers. Plant Cell Tissue Organ Cult. 2010;100:241–254. doi: 10.1007/s11240-009-9647-2. [DOI] [Google Scholar]
  8. Chand S, Singh AK. Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber-yielding leguminous tree species. J Plant Physiol. 2004;161:237–243. doi: 10.1078/0176-1617-01036. [DOI] [PubMed] [Google Scholar]
  9. Chand S, Singh AK. Plant regeneration from semi-mature zygotic embryos of Dalbergia sissoo Roxb. Indian J Biotech. 2005;4:78–81. [Google Scholar]
  10. Das P, Samantaray S, Roberts AV, Rout GR. In vitro somatic embryogenesis of Dalbergia sissoo Roxb.—a multipurpose timber yielding tree. Plant Cell Rep. 1997;16:578–582. doi: 10.1007/BF01142327. [DOI] [PubMed] [Google Scholar]
  11. Datta SK, Datta K. Auxin induced regeneration of forest trees-Dalbergia sissoo Roxb. through tissue culture. Curr Sci. 1983;52:435–436. [Google Scholar]
  12. Datta SK, Datta K, Paramanik T. In vitro clonal multiplication of mature tree of Dalbergia sissoo Roxb. Plant Cell Tissue Organ Cult. 1982;2:15–20. doi: 10.1007/BF00033548. [DOI] [Google Scholar]
  13. Dinkel-Meier A, Becker B, Duckstein D. Micropropagation and ex vitro rooting of several clones of flushing Quercus robur L. Ann Forest Sci. 1993;50:319–322. doi: 10.1051/forest:19930733. [DOI] [Google Scholar]
  14. Eeswara JP, Stuchbury T, Allan EJ, Mordue AJ. A standard procedure for the micropropagation of the Neem tree (Azadirachta indica A. Juss) Plant Cell Rep. 1998;17:215–219. doi: 10.1007/s002990050381. [DOI] [PubMed] [Google Scholar]
  15. Gulati A, Jaiwal PK. Micropropagation of Dalbergia sissoo from nodal explants of mature trees. Biol Plant. 1996;38:169–175. doi: 10.1007/BF02873840. [DOI] [Google Scholar]
  16. Gupta PK, Timmis R. Mass propagation of conifer trees in liquid cultures—progress towards commercialization. Plant Cell Tissue Organ Cult. 2005;81:339–346. doi: 10.1007/s11240-004-6654-1. [DOI] [Google Scholar]
  17. Huetteman CA, Preece JE. Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult. 1993;33:105–119. doi: 10.1007/BF01983223. [DOI] [Google Scholar]
  18. Husain MK, Anis M. Rapid in vitro multiplication of Melia azedarach L. a multipurpose woody tree. Acta Physiol Plant. 2009;31:765–772. doi: 10.1007/s11738-009-0290-7. [DOI] [Google Scholar]
  19. Husain MK, Anis M, Shahzad A. In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants. Acta Physiol Plant. 2008;30:353–359. doi: 10.1007/s11738-007-0130-6. [DOI] [Google Scholar]
  20. Ivanova M, Staden JV. Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoot of Aloe polyphylla. Plant Cell Tissue Organ Cult. 2008;92:227–231. doi: 10.1007/s11240-007-9311-7. [DOI] [Google Scholar]
  21. Joshi I, Bisht P, Sharma VK, Uniyal DP. Studies on effect of nutrient media for clonal propagation of superior phenotypes of Dalbergia sissoo Roxb. through tissue culture. Silvae Genet. 2003;52:143–147. [Google Scholar]
  22. Kim EK, Hahn EJ, Murthy HN, Paek KY. High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures. Plant Cell Tissue Organ Cult. 2003;73:231–236. doi: 10.1023/A:1023029302462. [DOI] [Google Scholar]
  23. Kopriva S, Mugford SG, Matthewman C, Koprivova A. Plant sulfate assimilation genes: redundancy versus specialization. Plant Cell Rep. 2009;28:1769–1780. doi: 10.1007/s00299-009-0793-0. [DOI] [PubMed] [Google Scholar]
  24. Kritikar KR, Basu BD (1975) Indian medicinal plants. Vol I New Delhi: M/S Periodical Experts, pp 818-819
  25. Lyod G, Mc Cown B. Commercially feasible micro propagation of Mountain Laurel Kalmia latifolia by use of shoot tip culture. Int Plant Prop Soc. 1981;30:421–427. [Google Scholar]
  26. Montenius O. In vitro meristem culture of juvenile and mature Sequoiandendron giagantium. Tree Physiol. 1987;3:265–272. doi: 10.1093/treephys/3.3.265. [DOI] [PubMed] [Google Scholar]
  27. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. [DOI] [Google Scholar]
  28. Pati PK, Kaur J, Singh P. A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don. Plant Cell Tissue Organ Cult. 2011;105:299–307. doi: 10.1007/s11240-010-9868-4. [DOI] [Google Scholar]
  29. Pattnaik S, Pradhan C, Naik SK, Chand PK. Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume Dalbergia sissoo. In Vitro Cell Dev Biol - Plant. 2000;36:407–411. doi: 10.1007/s11627-000-0073-8. [DOI] [Google Scholar]
  30. Pena L, Seguin A. Recent advances in the genetic transformation of trees. Trends Biotechnol. 2001;19:500–506. doi: 10.1016/S0167-7799(01)01815-7. [DOI] [PubMed] [Google Scholar]
  31. Peres ANA, Peres LEQ, Higashi EN, Martinelli AP. Adjustment of mineral elements in the culture medium for the micropropagation of three Vriesea bromeliads from the Brazilian Atlantic Forest: The Importance of Calcium. Hort Sci. 2009;44(1):106–112. [Google Scholar]
  32. Phulwaria M, Rai MK, Harish, Gupta AK, Ram K, Shekhawat NS (2012a) An improved micropropagation of Terminalia bellirica from nodal explants of mature tree. Acta Physiol Plant 34:299–305
  33. Phulwaria M, Ram K, Harish, Gupta AK, Shekhawat NS (2012b) Micropropagation of mature Terminalia catappa (Indian almond)—a medicinally important forest tree. J Forest Res 17:202–207
  34. Pijut MP, Beasley RR, Lawson SS, Palla KJ, Stevens EM, Wang Y. In vitro propagation of tropical hardwood tree species: a review. Propagat Ornament Plant. 2012;12:25–51. [Google Scholar]
  35. Pospisilova J, Ticha I, Kadleck P, Haisel D, Plzakova S. Acclimatization of micropropagated plants to ex vitro conditions. Biol Plant. 1999;42:481–497. doi: 10.1023/A:1002688208758. [DOI] [Google Scholar]
  36. Preece JE. Can nutrient salts partially substitute for plant growth regulators? Plant Tissue Cult Biotechnol. 1995;1:26–37. [Google Scholar]
  37. Preece JE. Micropropagation in stationary liquid media. Propagat Ornament Plant. 2010;10:183–187. [Google Scholar]
  38. Preece JE, Shutter EG (1991) Acclimatization of micropropagated plants to the greenhouse and field. In: Debergh PC, Zimmerman RH (eds) Micropropagation: Technology and application, pp 71–94
  39. Rai MK, Asthana P, Jaiswl VS, Jaiswal V. Biotechnological advances in Guava (Psidium guajava L.) recent developments and prospects for further research. Trees Struct Funct. 2010;24:1–12. doi: 10.1007/s00468-009-0384-2. [DOI] [Google Scholar]
  40. Rathore V, Shekhawat NS, Singh RP, Rathore JS, Dagla HR. Cloning of adult trees of jamun (Syzygium cuminii) Indian J Biotechnol. 2004;3:241–245. [Google Scholar]
  41. Sanjaya, Muthan B, Rathore TS, Rai VR (2006) Micropropagation of an endangered Indian sandalwood (Santalum album L.). J Forest Res 11:203–209
  42. Shekhawat NS, Rathore TS, Singh RP, Deora NS, Rao SR. Factors affecting in vitro cloning of Prosopis cineraria. Plant Growth Regul. 1993;12:273–280. doi: 10.1007/BF00027208. [DOI] [Google Scholar]
  43. Shekhawat NS, Singh RP, Deora NS, Kaul G, Kotwal RC, Choudhary N. Micropropagation of plants of stressed ecosystems. In: Shrivastava PS, editor. Plant Tissue Culture and Molecular Biology: Application and prospects. New Delhi: Narosa; 1998. pp. 579–586. [Google Scholar]
  44. Singh AK, Chand S. Somatic embryogenesis and efficient plant regeneration from cotyledon explants of a timber yielding leguminous tree Dalbergia sissoo Roxb. J Plant Physiol. 2003;160:415–421. doi: 10.1078/0176-1617-00523. [DOI] [PubMed] [Google Scholar]
  45. Singh AK, Chand S, Pattnaik S, Chand PK. Adventitious shoot organogenesis and plant regeneration from cotyledons of Dalbergia sissoo Roxb., a timber-yielding tree legume. Plant Cell Tissue Organ Cult. 2002;68:203–209. doi: 10.1023/A:1013870803937. [DOI] [Google Scholar]
  46. Thirunavoukkarasu M, Panda PK, Nayak P, Behera PR, Satpathy GB (2010) Effect of media type and explant source on micropropagation of Dalbergia sissoo Roxb. – an important multipurpose forest tree. Int Res J Plant Sci 1155-1162.
  47. Tripathi M, Kumari N. Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis. Acta Physiol Plant. 2010;32:1011–1015. doi: 10.1007/s11738-010-0484-z. [DOI] [Google Scholar]
  48. Yan H, Liang C, Yang L, Li Y (2010) In vitro and ex vitro rooting of Siratia grosvenori a traditional medicine plant. Acta Physiol Plant 32:115–120

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