In Vitro Cellular & Developmental Biology - Plant

, Volume 49, Issue 2, pp 216–222

Plant regeneration from leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Authors

    • Department of Agricultural BiotechnologyAnand Agricultural University
  • Sandeep R. Raj
    • Department of Agricultural BiotechnologyAnand Agricultural University
  • V. R. Patil
    • Department of Agricultural BiotechnologyAnand Agricultural University
  • P. S. Jaiswal
    • Department of Agricultural BiotechnologyAnand Agricultural University
  • N. Subhash
    • Department of Agricultural BiotechnologyAnand Agricultural University
Plant Tissue Culture

DOI: 10.1007/s11627-013-9495-y

Cite this article as:
Singh, C.K., Raj, S.R., Patil, V.R. et al. In Vitro Cell.Dev.Biol.-Plant (2013) 49: 216. doi:10.1007/s11627-013-9495-y

Abstract

Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg l−1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg l−1 TDZ. The WPM medium supplemented with 2.5 mg l−1 BA + 0.4 mg l−1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg l−1 BA and 3.0 mg 1−1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg l−1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.

Keywords

Callus inductionCallus frequencyShoot budSandalwoodAcclimatization

Introduction

Sandalwood (Santalum album) is a hemi-parasitic tree, occurring in semiarid areas from India to the South Pacific and the northern coast of Australia. In India, it is mostly found in the states of Andhra Pradesh, Karnataka, Tamil Nadu, Gujarat, Madhya Pradesh, and Maharashtra. Sandalwood from the Mysore region of Karnataka in Southern India is widely considered to be of the highest quality. It grows in geographical locations that receive 850–1,350 mm annual rainfall, with temperatures ranging from 25°C to 35°C (Rao et al.2007). The tree is medium sized, about 12–15 m tall, and reaches its full maturity in 60 to 80 yr. At maturity, the center of the trunk (the heartwood) achieves its greatest oil content. The oil is highly valued for its fragrance and is used in perfumes, cosmetics, and medical industries, among others. It is also a major constituent in agarbathi (incense sticks) manufacturing. S. album has the highest oil content (about 6%) among all the species of the genus Santalum.

This genus includes both trees and shrubs, the majority of which are non-obligate root parasites that photosynthesize their own carbohydrates, but access the roots of other species for water and inorganic nutrients. The best hosts for sandalwood are nitrogen-fixing trees because growth depends on the amino acid availability such that the host plant does not compete with the sandalwood for nutrients (Brand 2005).

Sandalwood is recognized worldwide as one of the most valuable commercial tree species with an estimated market value of more than $1 billion (Viswanath et al.2008). S. album is currently listed as “vulnerable” by the International Union of Conservation of Nature and Natural Resources of threatened species (IUCN 2012). There has been at least 20% loss over the last 10 yr or three generations, based on actual or potential levels of exploitation. The existing populations are devoid of trees of a commercial girth not only due to illicit felling, but also grazing, recurrent fires, and the lethal phytoplasmic spike epidemics. Natural regeneration of S. album is low because of low percentage of seed germination (10–20%), scavenging of germinated seeds by squirrels and rodents, as well as browsing and trampling of young seedlings by wildlife and cattle.

Conventional breeding of sandalwood for introgression of new genetically amicable traits is an expensive and difficult task because of their long generation time, sexual incompatibility, and heterozygous nature (Rugkhla 1997). For large-scale production of sandalwood, different in vitro techniques can be used to clone superior lines, which is a major prerequisite for Agrobacterium-mediated transformation and protoplast fusion gene transfer techniques. In vitro regeneration of S. album has been reported from hypocotyl, nodal, and endosperm explants (Bapat and Rao 1979; Lakshmi Sita et al.1979; Rao and Bapat 1992). Leaves from mature trees provide a useful source of explants, which are easily available and elicit a good response. Furthermore, simultaneous indirect plant regeneration via leaves will reduce the possibility of genetic variation common in plants regenerated from cultured immature cells or tissues. The first incidence of plant regeneration from callus using leaf explants from mature sandalwood trees is reported here. The objective of this study was to develop a more efficient and reliable protocol for in vitro regeneration of sandalwood from leaf explants of mature sandalwood trees using different combinations of plant growth regulators.

Material and Methods

Plant material and surface sterilization.

Young leaf explants about 10 cm2 were collected from wild species of mature trees of sandalwood (S. album L.). Single leaf samples were cut to ∼4 cm2 and dipped in 0.1% (v/v) liquid detergent (Tween-20) for 5 min. After this, explants were washed four or five times with distilled water. Explants were subsequently treated with 1,000 ppm Bavistin, 200 ppm cefotaxime, and 200 ppm kanamycin for 10 min and washed three times with sterile distilled water in a laminar flow hood. Subsequently, the explants were surface sterilized with 0.1% (w/v) mercuric chloride for 3–4 min and thoroughly washed six to eight times with sterile distilled water (Maina et al.2010). Following surface sterilization, all exposed ends of the explants were trimmed, and the remaining segment (∼1–1.5 cm) was inoculated horizontally on the culture medium.

Callus induction.

Surface-sterilized leaf explants (1–2 cm) were placed with the adaxial surface in contact with the callus induction media. The callus induction media were composed of basal woody plant medium (WPM) (Lloyd and McCown 1981) containing myo-inositol (100 mg l−1), K2SO4 (990 mg l−1), and sucrose (3%, w/v). These media were supplemented with different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 mg l−1) of thidiazuron (TDZ) and different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 mg l−1) of 2,4-dichlorophenoxyacetic acid (2,4-D). The pH of the medium was adjusted to 5.8 with 1 N NaOH before adding agar (0.8%, w/v). The same medium without plant growth regulators was used as a control. All cultures were incubated in a culture room maintained at 25 ± 2°C, 40–60% relative humidity, and 16/8-h light/dark regime under cool-white fluorescent light at 36 μmol m−2 s−1, except for cases where specific physiological conditions were required. The rate of callus formation was determined after 2 wk.

Fresh and dry weight of callus.

Growth measurements were made in terms of increase in the fresh and dry weights of cultured tissue. The cultured tissue grown on solid medium were carefully removed from the culture vessel and made free from specks of agar that had adhered at the point of contact. Accumulated water was blotted out gently without squeezing the tissue. The tissue was then transferred onto preweighed aluminum foil, and the weight was determined on a single pan digital balance. After recording the fresh weight, the tissues were oven-dried at 60°C to a constant weight on the same foils for determination of their dry weight.

Shoot bud induction and multiplication.

For shoot bud induction, the regenerated calli were transferred to the WPM medium supplemented with different combinations of BA (0.5, 1.0, 1.5, 2.0, or 2.5 mg l−1) and NAA (0.2 or 0.4 mg l−1). The frequency of shoot bud induction and number of shoot bud per calli were calculated after 8 wk. After shoot bud induction, calli were transferred to media supplemented with different concentrations of BA (1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) and Kn (1.0, 1.5, 2.0, 2.5, and 3.0 mg l−1) for the passage of shoot proliferation. The percentage of callus-producing shoots, number of shoots, and shoot length were recorded after 10 wk of culture.

Rooting and acclimatization.

For root induction, regenerated shoots (4–5 cm) were excised from the parent culture and transferred onto WPM medium supplemented with various concentrations of indole-3-butyric acid (IBA, 1.0, 1.5, 2.0, 2.5, or 3.0 mg l−1). Rooting percentages were recorded after 3 wk of root development. The in vitro-regenerated plantlets with well-developed shoots (3–4 cm) and roots were washed thoroughly in running tap water and transferred to plastic pots containing sterile soil and coco-peat potting mixture (1:1) for acclimatization in a greenhouse at 24 ± 2°C, with a 16-h photoperiod at a light intensity of 85 μmol m−2 s−1. Potted plantlets were first covered with transparent polythene bags to ensure maximum humidity. As the primary acclimatization of transferred explants was achieved, transparent covers were removed. During the process of primary and secondary hardening, the plants were watered every second day. Secondary hardening was carried out in a polyhouse to acclimatize in vitro-grown plants to natural physiological and environmental conditions.

Statistical analysis.

The experiment was conducted as a completely randomized design consisting of two factors of factorial arrangement. Each treatment consisted of a single explant per test tube (150×25 mm) and was replicated 12 times, representing a total of 12 observations per treatment. Data from all experiments were subject to ANOVA, mean comparison was analyzed by Duncan's multiple range test using SPSS (version 7.5), and the results were expressed as means ± SE.

Result and Discussion

Callus induction.

Callus was initiated from the cut ends of leaf sections on WPM media supplemented with different concentrations of TDZ or 2,4-D. After 2 wk, the entire surface of the explants was covered with callus. Both 2,4-D and TDZ were effective at inducing callus formation, but TDZ generated higher frequencies of callus than 2,4-D. In control media, no response was observed in comparison to treated media. Morphology and growth of the calli were affected by the concentration of growth regulators. Lower concentrations of TDZ and 2,4-D gave a better response as higher concentrations were toxic to the explants and caused browning. Lower concentrations of TDZ (0.4 and 0.6 mg l−1) showed the highest frequency of callus induction (Table 1). TDZ exhibits the unique property of mimicking both auxin and cytokinin effects on growth and differentiation of cultured explants, although structurally it is different from either auxins or purine-based cytokinins. TDZ at 0.2 mg l−1 showed less callus growth in leaf explants. Low concentrations of 2,4-D, i.e., 1.5 and 2.0 mg l−1 also showed a better response in comparison to higher concentrations. TDZ and 2,4-D were reported to induce callus formation in a variety of plant culture systems with a rate of cell proliferation and intrinsic activity higher than that obtained with other growth regulators (Capelle et al.1983; Murthy et al.1998), which are also in agreement with the present study. Huetteman and Preece (1993) also have reported that TDZ is a potent cytokinin for woody plant tissue culture.
Table 1

Effect of plant growth regulators on callus induction and its morphological characteristics derived from the leaf explant of S. album

Media WPM

Days to callus initiation

Callus frequency (%)

Callus growth

Callus morphology and nature

TDZ (mg l−1)

2,4-D (mg l−1)

0.2

0

8.65

41.66

++

W, F, and E

0.4

0

8.29

100

++++

Y, C, and E

0.6

0

8.22

91.6

++++

Y, C, and E

0.8

0

7.92

83.33

+++

W, F, and NE

1.0

0

8.60

58.33

+++

W, F, and NE

1.2

0

8.67

33.33

+++

Y, C, and E

0

0.5

8.35

50

++

Y, C, and E

0

1.0

8.40

58.33

++

Y, C, and NE

0

1.5

8.33

75

+++

Y, C, and E

0

2.0

8.5

66.66

+++

W, F, and NE

0

2.5

8.45

50

++

Y, C, and NE

0

3.0

8.70

25

+

W, F, and NE

0

0

0

0

W white, Y yellow, F friable, C compact, E embryogenic, NE non-embryogenic, ++++ excellent, +++ good, ++ average, + poor, − no response

Callus growth and morphology.

Lower concentrations of TDZ at 0.4 and 0.6 mg l−1 showed excellent callus growth from leaf explants in comparison to higher concentrations of growth regulators (Fig. 1ac; Table 1). Lower concentrations of 2,4-D, 1.5 and 2.0 mg l−1, also showed good callus growth. TDZ and 2,4-D at low concentration stimulate cell division by encouraging the synthesis of endogenous purines and cytokinins, and inhibit their degradation (Thomas and Katterman 1986). The leaf explant-derived callus under different levels of TDZ and 2,4-D showed both yellow and white coloration with compact and embryogenic-type cells (Fig. 1c). Our results showed that lower concentrations of TDZ and 2,4-D are generally inclined to promote the formation of a compact yellow nodular callus. At higher concentrations, the callus was white and friable initially and later turned into a compact nodular callus after prolonged incubation. This is in agreement with the observations of Sahai et al. (2010) where slightly compact yellowish green calli were produced from the cut ends of leaf segments of Tylophora indica on medium with low concentrations of TDZ.
https://static-content.springer.com/image/art%3A10.1007%2Fs11627-013-9495-y/MediaObjects/11627_2013_9495_Fig1_HTML.gif
Figure 1

In vitro regeneration from leaf explants of S. album. (a) Leaf section from a mature S. album tree on media. (b) Callus induction from a leaf section on WPM medium with 0.4 mg l−1 after 2 wk. (c) Proliferated callus from a leaf section after 6 wk. (d) Shoot bud induction from leaf-derived callus after 14 d. (e) Microscopic view of callus showing developing shoot bud primordia (arrow). (f) Shoot bud proliferation from leaf-derived callus after 28 d. (g) Regenerated shoot from leaf-derived callus through indirect organogenesis. (h) Root initiation from an in vitro shoot. (i) Hardened in vitro-regenerated plant of S. album for acclimatization.

Fresh and dry weight.

Leaf-derived callus at low-mid-range concentrations of TDZ and 2,4-D showed maximum fresh weight and dry weight in media (Table 2). A lower concentration of 2,4-D, i.e., 2.0 mg l−1 showed the highest fresh weight (133.4 mg) and dry weight (38.3 mg) of callus, whereas the medium supplemented with TDZ at 0.8 mg l−1 showed the highest fresh weight (141.9 mg) and dry weight (47 mg). Similar observations were also made by Wang et al. (2011), where fresh weight of Rheum franzenbachii callus grown on medium supplemented with TDZ was higher compared to other treatments, and dry weight of callus was highest on medium supplemented with 0.5 mg l−1 TDZ was increased. The above outcomes are in accordance with the findings of Janarthana and Seshadri (2008) and Mungole et al. (2011) where maximum fresh weight of callus was obtained on medium supplemented with 2,4-D.
Table 2

Effect of different plant growth regulators on callus proliferation from leaf explants of S. album after 6 wk

Media WPM

Fresh weight of callus (g)a

Dry weight of callus (g)a

% of dry matterb

TDZ (mg l−1)

2,4-D (mg l−1)

0.2

0

113.5 ± 6.0f

16 ± 3.1h

14.55 (3.84)

0.4

0

134.8 ± 5.7c

30.8 ± 6.2e

22.54 (4.80)

0.6

0

138.4 ± 5.5b

42.8 ± 6.2b

30.75 (5.59)

0.8

0

141.9 ± 5.5a

47.0 ± 6.0a

33.02 (5.79)

1.0

0

130.6 ± 5.5e

31.3 ± 3.6e

23.80 (4.93)

1.2

0

129.5 ± 4.8ef

31.3 ± 2.8e

24.10 (4.96)

0

0.5

130.3 ± 5.2e

32.3 ± 2.3e

24.70 (5.02)

0

1.0

128.1 ± 5.1ef

33.4 ± 3.6de

26.02 (5.15)

0

1.5

132.0 ± 3.8cd

36.5 ± 2.1cd

27.59 (5.30)

0

2.0

133.4 ± 3.6cd

38.3 ± 2.3c

28.55 (5.39)

0

2.5

128.9 ± 4.2ef

28.3 ± 4.6f

21.87 (4.73)

0

3.0

129.7 ± 3.6ef

25.7 ± 3.4g

19.66 (4.49)

0

0

30.3 ± 5.0g

6.2 ± 1.9i

20.38 (4.47)

aValues represent mean ± SE. Means in each column followed by the same letter are not significantly different according to DMRT at α = 0.05

bData transferred through arc sine transformation. Values in parentheses indicate original mean values

Similar to the media producing the highest fresh and dry weight of calli, the percent of dry matter in the presence of TDZ, i.e., 0.8 mg l−1, and 2,4-D, i.e., 2.0 mg l−1, showed the highest dry matter percentages, 33.02 and 28.55, respectively (Table 2).

Shoot bud induction.

Morphogenic callus, when subcultured onto WPM medium supplemented with different combinations and concentrations of BA and NAA, gave rise to greenish, nodular, and more organized differentiated tissues (Fig. 1d). De novo shoot buds differentiated from the surface of callus with distinct visible leaf primordia (Fig. 1e). Among various combinations and concentrations of BA and NAA tested, the maximum shoot bud frequency (91.66%) and number of shoot buds per callus (24.66 ± 2.81) were obtained at 2.5 mg l−1 BA + 0.4 mg l−1 NAA (Table 3; Fig. 1f). A high cytokinin-to-auxin ratio usually results in shoot induction (Krikorian 1995). Azad et al. (2005) and Chandra and Bhanja (2002) also noted the maximum number of adventitious shoots regenerated from the leaf-derived callus of woody species supplemented with BAP, which is in congruence with the present study.
Table 3

Effect of different combinations of BA and NAA on shoot bud induction from leaf-derived callus

Media WPM

Number of shoot buds per callusa

Bud frequency (%)

BA (mg l−1)

NAA (mg l−1)

0.5

0.2

14.91 ± 2.15c

50.00

1.0

0.2

15.16 ± 2.12c

58.33

1.5

0.2

20.25 ± 1.96bc

66.66

2.0

0.2

22.08 ± 1.62b

75.00

2.5

0.2

24 ± 3.46ab

79.16

0.5

0.4

16.5 ± 2.81c

58.33

1.0

0.4

17.08 ± 3.12c

70.83

1.5

0.4

22.83 ± 1.99b

66.66

2.0

0.4

23.75 ± 2.14b

75.00

2.5

0.4

24.66 ± 2.81a

91.66

0

0

0d

0

aValues represent means ± SE. Means in each column followed by the same letters are not significantly different according to DMRT at α = 0.05

Shoot proliferation and differentiation.

In vitro shoots were regenerated to complete plantlets when they were cultured in WPM media containing a certain level of cytokinins. Among various concentrations of BA and Kn tested, the maximum shoot regeneration frequency (91.60%) and number of shoots (20.67 ± 2.7) were obtained with 5.0 mg l−1 BA. However, maximum shoot length (5.17 ± 0.6 cm) was obtained at 3.0 mg l−1 Kn (Table 4; Fig. 1g). Due to the diverse biochemical and morphological responses elicited in plants by kinetin, it was of interest to learn whether a compound such as kinetin is directly concerned in promoting cell division, as is now generally assumed, or whether this compound serves merely to trigger in planta synthesis of substances that are specifically involved in that process (Wood and Braun 1967). The results of the present study agree with Vyas et al. (2005), where the maximum number of adventitious shoots was regenerated from leaf-derived callus supplemented with BAP and Kn.
Table 4

Effect of different cytokinins at different levels on shoot proliferation from leaf-derived callus

WPM

Shoot percentage per explants

Number of shoots per explant1

Shoot length (cm)a

BA (mg l−1)

Kn (mg l−1)

1.0

0

58.33

14.17 ± 1.6b

3.17 ± 0.9c

2.0

0

54.16

14.5 ± 1.2b

3.58 ± 1.0c

3.0

0

62.50

17.83 ± 2.6b

3.83 ± 0.7c

4.0

0

70.83

19.5 ± 3.1a

4.50 ± 0.5b

5.0

0

91.60

20.67 ± 2.7a

5.00 ± 0.6ab

0

1.0

54.16

16.5 ± 2.8b

4.00 ± 0.7bc

0

1.5

66.66

17.08 ± 3.1b

3.92 ± 0.8c

0

2.0

62.50

19.33 ± 3.8ab

4.00 ± 1.0b

0

2.5

70.83

19.67 ± 3.1a

4.75 ± 0.5b

0

3.0

79.16

19.92 ± 2.8a

5.17 ± 0.6a

0

0

50.00

3.58 ± 0.7c

1.75 ± 0.6d

aValues represent means ± SE. Means in each column followed by the same letter are not significantly different according to DMRT at α = 0.05

Rooting and acclimatization.

The regenerated shoots produced roots when transferred to media supplemented with various concentrations of IBA. The percentage of in vitro shoots that rooted significantly differed depending upon the concentration of IBA in the medium. The best rooting percent (91.67) was observed when elongated shoots were cultured with WPM medium containing 1.5 mg l−1 IBA (Table 5; Fig. 1h). The findings on percentage of rooting are in agreement with the results of Ali et al. (2009) who concluded that IBA proved to be the best auxin for rooting in olives and with Metivier et al. (2007) where IBA at 10 μM proved to be the most suitable treatment for 100% rooting in the woody species Cotinus coggygria. Tissue culture-raised plantlets were transferred from in vitro to in vivo conditions for hardening. For proper acclimatization, plantlets were kept under controlled environmental conditions in a greenhouse.
Table 5

Effect of different concentrations of IBA on root initiation from regenerated shoots of S. album

Media WPM

Rooting percentage (%)

IBA (mg l−1)

0

41.67

1.0

66.67

1.5

91.67

2.0

58.33

2.5

75.00

3.0

50.00

After 4–5 wk, more than 90% of plants were able to survive on the substrate made of coco-peat and soil (1:1; Fig. 1i). Shukla and Mishra (2009) achieved more than 80% plant survival and growth in the medicinal tree Stereospermum personatum, when tissue culture-raised plantlets were transferred to net pots containing coco-peat for acclimatization in the greenhouse. Similar results were also obtained in the present investigation in sandalwood proving coco-peat as a good hardening medium for in vitro-raised plantlets of this woody species.

These results demonstrate that leaf explants of S. album have significant morphogenetic potential for callus formation and indirect organogenesis; however, the response is highly sensitive and directly related to the combinations of exogenous growth regulators in the culture medium. A reliable in vitro regeneration technique is an essential component for most methods of genetic transformation (Schwarz and Beaty 2000) and protoplast fusion.

Conclusions

In the present investigation, the protocol for the in vitro regeneration from leaf explants has been developed. This is simple, economical, rapid, and highly reproducible to obtain more plantlets within a short period. Regeneration via callus has been a potent source of producing parental clones as well as somaclonal variants in plants. Moreover, regeneration involving a callus phase would be suitable for combining transformation events to recover transgenic plants. Hence, the above procedure may be employed in relation to the aforesaid facts for genetic improvement and also could be used as a tool to introduce new variants of S. album in a somaclonal variant selection program.

Acknowledgments

The authors gratefully acknowledge the Plant Tissue Culture Laboratory, Department of Agricultural Biotechnology, Anand Agricultural University, Anand, Gujarat, India, for providing laboratory facilities.

Copyright information

© The Society for In Vitro Biology 2013