Plant Growth Regulation

, Volume 68, Issue 2, pp 293–301

Effect of plant growth regulators on plant regeneration of Dioscorea remotiflora (Kunth) through nodal explants

Authors

  • A. Bernabé-Antonio
    • Department of Agricultural ProductionUniversity Center of Biological and Agricultural Sciences, University of Guadalajara
    • Department of Agricultural ProductionUniversity Center of Biological and Agricultural Sciences, University of Guadalajara
  • F. Cruz-Sosa
    • Department of Biotechnology, Division of Biological and Health SciencesMetropolitan Autonomous University-Iztapalapa Campus
Original paper

DOI: 10.1007/s10725-012-9717-z

Cite this article as:
Bernabé-Antonio, A., Santacruz-Ruvalcaba, F. & Cruz-Sosa, F. Plant Growth Regul (2012) 68: 293. doi:10.1007/s10725-012-9717-z

Abstract

Dioscorea remotiflora (Kunth) is an important wild plant that produces tuberous roots used as a source of food in the Western part of Mexico. Lack of planting material and inefficiency of traditional methods of propagation are the main constraints for implementing large-scale cultivation. In contrast, tissue culture techniques allow increasing multiplication and rapid production of plant material. In this regard, leaves or nodal segments were incubated on MS, B5 and WPM culture media with different PGRs in order to obtain an efficient micropropagation protocol. Leaves explants were unable to inducing shoots or callus. However, nodal segments produced axillary shoots and/or callus in all culture media. MS containing 2.33 μM KIN was the most suitable to inducing shoots; an average of 6.6 shoots per segment for 100 % explants was obtained, which displayed also the greater number of nodes (5.0) and leaves (7.9) per segment. A decrease on shoot proliferation was observed combining BA or KIN with 2,4-D or NAA. However, small brownish callus were induced on 100 % of segments using 2.33 μM KIN with 5.37 μM 2,4-D or 9.30 μM KIN plus 2.69 μM NAA. In contrast, by adding 2.69 μM NAA, 66.4 % of the nodal segments formed shoots and produced also yellowish friable callus on the base of the shoots. Shoots were easily rooted with 8.28 μM IBA (96.9 %), displaying the greatest root and shoot biomass, but maximum number of tuberous roots, and root or tuberous root biomass was produced increasing IBA (20.7 μM).

Keywords

Tissue cultureCulture mediaMicropropagationDry weightTuberous roots

Abbreviations

ANOVA

Analysis of variance

BA

N6-Benzyladenine

DW

Dry weight

IBA

Indole-3-butyric acid

PGRs

Plant growth regulators

PVP

Polyvinylpyrrolidone

KIN

Kinetin

NAA

α-Naphthaleneacetic acid

WPM

Woody plant medium

2,4-D

2,4-Dichlorophenoxyacetic acid

Introduction

The genus Dioscorea is a monocotyledonous plant belonging to the Dioscoreaceae family and comprises 600 species approximately, mainly distributed in subtropical and temperate areas of Africa, India, Southeast Asia, Australia and America (Ayensu 1972). In the case of Mexico, D. remotiflora Kunth is a wild plant known popularly as “camote de cerro”, mainly distributed in the Pacific and Center Zone of the country (González 1984). Because these species are not cultivated, tubers from wild plants are collected by residents of some localities and used as a source of food containing high nutritional value (Guízar-Miranda et al. 2008). However, this has caused a decline in wild plants which could be avoided by using plant tissue culture technique, a plant biotechnology tool that provides us with resources to develop protocols for mass micropropagation of healthy plants and use them to establish extensive crops.

The main problem in Dioscorea species cultivation for commercial purposes is the use of traditional protocols deficient on sexual and asexual propagation (Forsyth and Staden 1982). In this regard, some studies have been aimed to develop micropropagation and microtuberization yams protocols. However, responses of explants have been affected by external and internal factors, i.e., plant growth regulators, development phase of the mother plant, type and size of explant and genotype (Staba 1982). In fact, it was found that the addition of 2 mg l−1 KIN could reduce multiplication rate of some clones in D. cayenensisD. rotundata (Ondo et al. 2007). Furthermore, Viana and Mantell (1989) found that regeneration of plantlets from D. cayenensis calluses occurred only at low levels of 2,4-D (0.5 mg l−1) and there were genotype-dependent differences between yam species in their ability to regenerate plantlets in vitro. Other wild species of Dioscorea have been also examined, including D. composita (Alizadeh et al. 1998), D. alata (Borges et al. 2004; Vaillant et al. 2005), D. oppositifolia (Behera et al. 2009), D. zingiberensis (Chen et al. 2003; Huang et al. 2009; Yuan et al. 2005), D. nipponica (Chen et al. 2007), D. polystachya, D. sansibarensis and D. japonica (Islam et al. 2008), and D. opposita (Kohmura et al. 1995; Xu et al. 2009). To our knowledge, however, there are no reports about micropropagation of D. remotiflora Kunth.

The aim of this study is to evaluate different combinations and concentrations of some plant growth regulators (PGRs) on different culture media to obtain an efficient micropropagation protocol of D. remotiflora Kunth via nodal segments.

Materials and methods

Plant material

Tubers of wild plants were collected in September 2007 in Acatic, State of Jalisco, Mexico. A voucher was registered as # 68191 at the herbarium of Universidad Autónoma Metropolitana-Campus Iztapalapa (UAM-I). Tubers collected were disinfected for 5 min in 0.1 % (v/v) triclorometiltio-4-ciclohexano-1,2-dicarboximida (Captan Ultra 50WR) solution and then dried for 1 day at room temperature. Tubers were placed in paper bags and they were stored at 25 ± 2 °C. After storing them for 4 months tubers sprouted. Then, shoots of 5–10 cm length were excised and used as source of plant material for in vitro establishment. Shoots were disinfected superficially using a soap solution for 5 min, followed by immersion in a 0.05 % (v/v) d-Alanine, N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-methyl ester (Ridomil Gold 480 SL) solution, for 10 min. With low and constant agitation, shoots were then immersed for 10 min into a 1.8 % (v/v) sodium hypochlorite solution with one drop of liquid soap per 100 ml of prepared solution added. Under aseptic conditions, shoots were rinsed three times with sterilized distilled water. Disinfected shoots were transferred to Petri dishes and nodal segments of 1 cm length were cut. Two nodal segments were placed per jar containing 25 ml of culture medium. MS (Murashige and Skoog 1962) culture medium supplemented with L2 vitamins (Phillips and Collins 1979), sucrose 3 %, 0.8 % (w/v) agar and 9.3 μM KIN were used for in vitro maintenance (Ondo et al. 2007). Axillary shoot proliferation was maintained by subculturing single-node leafy cuttings every 2 or 3 months for 1 year on the same medium. Leaves and nodal segments were used as a source of explants for the subsequent micropropagation experiments.

Culture media and incubation conditions

Leaves or nodal segments of D. remotiflora were used to evaluate the ability of different culture media to support shoot induction on MS, B5 (Gamborg et al. 1968) and WPM (Lloyd and McCown 1980) adding 3, 2 and 2 % (w/v) sucrose, respectively. Citric acid (100 mg l−1), ascorbic acid (150 mg l−1) and polyvinylpyrrolidone (PVP; Sigma-Aldrich, St. Louis, MO, USA) (250 mg l−1) were also added to culture media to avoid necrosis of explants. In order to examine the effect of plant growth regulators (PGRs) on shoot proliferation, basal media were supplemented with 0, 2.33, 4.65 or 9.30 μM of BA or KIN combined with 0, 2.69, 5.37 or 10.74 μM of NAA or 2,4-D. All cultures were solidified with 0.2 % (w/v) phytagel (Sigma-Aldrich, St. Louis, MO, USA), adjusted to pH 5.8 ± 0.1, prior to autoclaving at 121 °C for 18 min. Leaves were cut into 5 mm × 5 mm segments, and nodal segments of about 1.5 cm length were also cut and they were immersed into an antioxidant solution with citric acid 100 mg l−1 plus ascorbic acid 150 mg l−1 for 5 min. Leaves or nodal segments were horizontally placed into glass jars containing 25 ml of culture medium. Four glass jars with four segments per jar were used for each treatment and the experiment was repeated twice. In a controlled environment growth chamber, cultures were incubated at 25 ± 1 °C and 60 μmol m−2 s−1 photosynthetic photon flux density under a 16 h illumination cycle. They were maintained by regular subcultures at 2 week intervals on fresh medium with the same compositions for 4 months. For each treatment, data of percentage of shoot formation, callus induction, simultaneous formation of shoots and callus, number of shoots, number of nodes, number of leaves, and length of shoot (cm) were recorded at 35 days of culture.

Four months old axillary shoots showing the best response in terms of percentage of shoot induction, mean number of shoots and mean number of nodes per segment were selected as a source of explants for the following experiments using different concentrations of IBA (0.04, 0.41, 4.14, 8.28, 20.70 and 41.40 μM) to evaluate rooting ability and formation of tuberous roots. Each treatment was carried out with four glass jars containing four segments per jar and the experiment was repeated twice. In all in vitro culture treated with IBA, mean number of tuberous roots, and dry biomass of plantlet (foliage, roots or tuberous roots) were evaluated as another parameter of grow, measured at 90 days of culture. The shoots, roots and tuberous roots were removed and dried in an oven (Terlab MA H45DM) at 60 °C for 72 h. Data were registered as milligrams (mg) of dry weight (DW).

Statistical analysis

SAS 9.0 software (SAS Institute Inc, 2002) was used for statistical analysis. Data of percentages of shoot induction, mean number of shoots per segment, means number of nodes per segment, means number of leaves per segment, means number of roots or tuberous roots per segment, mean length of shoot, and mean weight of dry biomass (foliage, roots or tuberous roots) were subjected to an analysis of variance (ANOVA), followed by Duncan multiple media comparison test. P-values <0.05 were considered significant.

Results and discussion

Axillary shoot induction

After 3 months, leaves segments were unable to induce shoots or callus using PGRs (BA, KIN, NAA and 2,4-D), concentrations ranged from 0 to 10.74 μM. However, similar work carried out in D. opposita and D. zingiberensis showed efficient shoot induction using leaves (Kohmura et al. 1995; Chen et al. 2003; Yuan et al. 2005). Many factors are known to influence the proliferation response in vitro. These may include the presence or absence of growth regulators either singly or in combination, type of explant, and genotype (Ondo et al. 2007; Staba 1982). However, when nodal segments were used, a rapid response of induction was observed on the three different basal media (MS, B5 and WPM). All variables were influenced by different basal media, showing statistically significant differences (P ≤ 0.05), except for callus induction (Table 1). Nodal segments displayed visible growth of shoots at axillary bud sites in all culture media types at 3 weeks of culture. Shoots induced by the different treatments were single in some instances and multiple in others. Simultaneously, callus formation was also observed on the shoots base. B5 and WPM culture media produced the lowest responses. In contrast, MS medium supported the best results for percentage of explants response, showing 37.4 % of shoot induction, 32.6 % of callus induction and 20.8 % of simultaneous formation of shoots and callus (Table 1). In addition, MS medium formed the highest mean number of shoots (2.0), mean number of nodes (2.9) and mean number of leaves per segment (5.2), as well as mean length of shoot (2.4 cm) (Table 1). Because responses induced using WPM and B5 media were inefficient, only the results produced with PGRs treatments on MS medium are shown.
Table 1

Effects of different culture media on shoots and/or callus induction of D. remotiflora Kunth nodal segments at 35 days of culture

Culture media

Induction (%)

Shoots/segment

Nodes/segment

Leaves/segment

Shoot length (cm)

Shoots

Callus

Shoots and callus

MS

37.4 ± 0.1a

32.6 ± 1.6a

20.8 ± 1.0a

2.0 ± 0.0a

2.9 ± 0.0a

5.2 ± 0.2a

2.4 ± 0.0a

B5

33.1 ± 2.3a

29.0 ± 3.9a

19.8 ± 2.1a

1.5 ± 0.2b

1.2 ± 0.1b

1.9 ± 0.3b

1.9 ± 0.3b

WPM

27.1 ± 4.2b

33.8 ± 3.9a

9.8 ± 2.0b

1.1 ± 0.4c

1.0 ± 0.5b

1.4 ± 0.5b

1.5 ± 0.2c

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

When the effects of different PGRs on nodal segments were evaluated using MS medium, it was found that individual addition of BA or KIN did not show significant differences (P ≤ 0.05) on shoot and/or callus induction (Table 2). However, KIN alone was the more suitable component producing 100.0 % of shoot formation in all three concentrations. Furthermore, the highest mean number of shoots (6.6), mean number of nodes (5.0), and mean number of leaves per segment (7.9) were obtained using KIN 2.33 μM (Table 2, Fig. 1a). Maximum mean length of shoot per segment (4.4 cm) was displayed with KIN 9.30 μM alone (Table 2). Both cytokinins, BA and KIN were unable to induce callus formation. It is known that cytokinins play important roles in various processes during growth and development of plants, including promotion of cell division, inhibition of senescence, regulation of apical dominance, and transmission of nutritional signals (Sakakibara 2010). However, tissue responses depend on plant species, as well as on tissue and developmental stage (Staba 1982). In this regard, some studies have reported that addition of 2.0 mg l−1 KIN to culture media reduces multiplication rate (node number) of certain D. cayensis clones (Ondo et al. 2010). Forsyth and Staden (1982) reported low responses of shoot formation in nodal segments of D.bulbifera using KIN 0.5 mg l−1, but number of shoots per segment were increased from 5 to 8 when KIN concentrations were raised from 5.0 to 10.0 mg l−1.
Table 2

Effects of BA or KIN on shoots and/or callus induction of D. remotiflora Kunth nodal segments at 35 days of culture on MS medium

PGRs (μM)

Induction (%)

Shoots/segment

Nodes/segment

Leaves/segment

Shoot length (cm)

BA

KIN

Shoots

Callus

Shoots and callus

0.00

0

96.2 ± 5.4a

0.0 ± 0.0a

0.0 ± 0.0a

4.1 ± 0.2c

3.1 ± 0.2b

3.8 ± 0.2c

4.1 ± 0.0b

2.33

0

100.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

3.4 ± 0.2d

1.1 ± 0.1d

2.3 ± 0.1d

2.6 ± 0.1d

4.65

0

100.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

3.3 ± 0.1d

1.0 ± 0.0d

0.9 ± 0.1e

2.7 ± 0.1d

9.30

0

93.8 ± 8.8a

0.0 ± 0.0a

0.0 ± 0.0a

2.0 ± 0.1e

0.5 ± 0.0e

0.9 ± 0.1e

2.3 ± 0.1e

0

2.33

100.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

6.6 ± 0.2a

5.0 ± 0.0a

7.9 ± 0.5a

3.9 ± 0.2b

0

4.65

100.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

4.5 ± 0.1b

3.3 ± 0.2b

5.3 ± 0.4b

3.3 ± 0.0c

0

9.30

100.0 ± 0.0a

0.0 ± 0.0a

0.0 ± 0.0a

4.8 ± 0.2b

2.3 ± 0.1c

4.8 ± 0.4b

4.4 ± 0.0a

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

https://static-content.springer.com/image/art%3A10.1007%2Fs10725-012-9717-z/MediaObjects/10725_2012_9717_Fig1_HTML.jpg
Fig. 1

Shoot, root and callus responses induced in nodal explants of D. remotiflora Kunth under several PGRs treatments on MS medium: a shoots proliferation with 2.33 μM KIN at 4 weeks of culture; b simultaneous formation of shoots and callus at 4 weeks of culture with 2.69 or 5.44 μM NAA; c callus excised from b after 1 month of culture; d shoots formation and spongy callus using combinations of BA with 2,4-D or NAA; e rooted plantlet with 8.28 μM IBA at 35 days of culture, and f plantlets with 8.28 μM IBA after 90 days of culture used to measure dry biomass. Scale bar = 1 cm

On the other hand, when 2,4-D or NAA auxins alone were used, the results were different, these were able to induce formation of shoots and/or callus (Table 3, Fig. 1b). Culture medium containing 2,4-D (5.37 μM) produced better percentages of shoot induction (25.0 %) compared with NAA (Table 3). However, this treatment produced the lowest mean number of shoots (1.4), mean number of nodes (0.1) and mean number of leaves per segment (0.4). Moreover, shoots produced were malformed and had a twisted appearance, whilst induced callus were friable, dark brown in color and a lack of growth in all three levels of 2,4-D was observed. Malformed shoots might be due to 2,4-D auxin that usually inhibits organogenesis and results to callus formation (Staba 1982). Medium containing 5.37 μM NAA displayed the best results in terms of callus induction (38.1 %), mean number of shoots (4.4) and mean number of leaves per segment (5.0). Whereas, the largest simultaneous formation of shoots and callus (66.4 %), mean number of nodes per segment (3.5) and mean length of shoot (4.5 cm) were produced using NAA 2.69 μM (Table 3, Fig. 1b), indicating that addition of auxin may also stimulate growth of plantlets (Pierik 1987). A larger number of nodes per segment was also produced in D. composita using NAA 2.5 μM (Alizadeh et al. 1998). Callus obtained using 2.69 or 5.37 μM NAA were friable; therefore, they were transferred to new culture medium, but a poor increase in the size of callus was observed at 2 weeks after transfer to fresh medium (Fig. 1c).
Table 3

Effects of 2,4-D or NAA on shoots and/or callus induction of D. remotiflora Kunth nodal segments at 35 days of culture on MS medium

PGRs (μM)

Induction (%)

Shoots/segment

Nodes/segment

Leaves/segment

Shoot length (cm)

2,4-D

NAA

Shoots

Callus

Shoots and callus

2.69

0

6.7 ± 1.9c

12.5 ± 0.2d

64.6 ± 3.0a

2.5 ± 0.2d

0.5 ± 0.0d

1.5 ± 0.1c

1.9 ± 0.1f

5.37

0

25.0 ± 0.0a

31.3 ± 2.5b

37.5 ± 6.4c

1.4 ± 0.0e

0.1 ± 0.0e

0.4 ± 0.0d

4.0 ± 0.0b

10.74

0

22.3 ± 0.6ab

15.4 ± 2.3d

37.5 ± 6.4c

1.5 ± 0.2e

0.5 ± 0.1d

0.5 ± 0.1d

3.0 ± 0.0d

0

2.69

19.0 ± 1.4bc

14.6 ± 0.4d

66.4 ± 7.4a

4.0 ± 0.1b

3.5 ± 0.2a

4.9 ± 0.2a

4.5 ± 0.1a

0

5.37

14.6 ± 3.0c

38.1 ± 3.1a

47.3 ± 6.1bc

4.4 ± 0.1a

3.1 ± 0.1b

5.0 ± 0.4a

3.4 ± 0.1c

0

10.74

22.5 ± 3.5ab

22.5 ± 3.5c

55.0 ± 7.1ab

2.9 ± 0.2c

0.9 ± 0.1c

2.0 ± 0.1b

2.4 ± 0.1e

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

When nodal segments were incubated in the presence of BA along with 2,4-D or NAA results showed highly significant differences (P ≤ 0.05) in all variables, showing the highest shoot induction (87.5 %) with 9.30 μM BA and 2.69 μM 2,4-D, but callus formation was not induced with the same concentrations (Table 4). Furthermore, the highest percentage of callus induction (75.0 %) was obtained when nodal segments were incubated either with BA (4.65 or 9.30 μM) along with 2,4-D (5.37 or 10.74 μM) or using 2.33 μM BA with 10.74 μM 2,4-D (Table 4); however, callus were small and browning. Combinations used with BA along with NAA showed a decrease in the percentage of callus induction in ranges from 25.0 to 62.5 %, and displayed a hard or spongy callus appearance, and at least one or two shoots per segment (Fig. 1d). Moreover, response frequency of shoot induction was higher compared with BA and 2,4-D (Table 4). Additionally, low concentrations of BA, 2.33 μM combined with NAA (2.69, 5.37 or 10.74 μM) produced the highest mean number of leaves (1.3), mean number of nodes (0.75), and simultaneous formation of shoots and callus (50.0 %), respectively; whereas, a mean of 2.0 shoots per segment was induced using 4.65 μM BA with 2.69 μM NAA (Table 4). Only 17.7 % of treatments produced simultaneous formation of shoots and callus when BA was combined with 2,4-D or NAA. Nevertheless, similar studies have shown positive effects on shoot induction of D. zingiberensis with BA and low concentrations of NAA, but shoot elongation was significantly inhibited (Chen et al. 2003). In another work carried out on nodal segments of D. oppositifolia, a high proliferation of shoots using two cytokinins at once (KIN and BA) combined with NAA was observed (Behera et al. 2009).
Table 4

Effects of BA and 2,4-D or NAA on shoots and/or callus induction of D. remotiflora Kunth nodal segments at 35 days of culture on MS medium

PGRs (μM)

Induction (%)

Shoots/segment

Nodes/segment

Leaves/segment

Shoot length (cm)

BAP

2,4-D

NAA

Shoots

Callus

Shoots and callus

2.33

2.69

0

37.5 ± 17.7de

62.5 ± 17.7ab

0.0 ± 0.0c

1.5 ± 0.0e

0.3 ± 0.0c

0.3 ± 0.0h

3.5 ± 0.3a

2.33

5.37

0

0.0 ± 0.0g

25.0 ± 0.0e

25.0 ± 0.0b

1.3 ± 0.0g

0.3 ± 0.0c

0.4 ± 0.0g

2.3 ± 0.0cd

2.33

10.74

0

0.0 ± 0.0g

75.0 ± 0.0a

0.0 ± 0.0c

0.0 ± 0.0h

0.0 ± 0.0d

0.0 ± 0.0i

0.0 ± 0.0g

4.65

2.69

0

29.2 ± 5.9ef

58.3 ± 11.8abc

0.0 ± 0.0c

1.5 ± 0.0g

0.3 ± 0.0c

0.4 ± 0.0g

2.6 ± 0.0b

4.65

5.37

0

25.0 ± 11.7ef

75.0 ± 11.7a

0.0 ± 0.0c

1.8 ± 0.1c

0.3 ± 0.0c

0.5 ± 0.1f

1.8 ± 0.1e

4.65

10.74

0

12.5 ± 5.8 fg

75.0 ± 11.7a

0.0 ± 0.0c

0.0 ± 0.0h

0.0 ± 0.0c

0.0 ± 0.0i

0.0 ± 0.0g

9.30

2.69

0

 

87.5 ± 5.9a

0.0 ± 0.0f

0.0 ± 0.0c

1.9 ± 0.0b

0.0 ± 0.0c

0.6 ± 0.1e

2.3 ± 0.1cd

9.30

5.37

0

0.0 ± 0.0g

75.0 ± 11.7a

0.0 ± 0.0c

0.0 ± 0.0h

0.0 ± 0.0c

0.0 ± 0.0i

0.0 ± 0.0g

9.30

10.74

0

0.0 ± 0.0g

75.0 ± 11.7a

0.0 ± 0.0c

0.0 ± 0.0h

0.0 ± 0.0c

0.0 ± 0.0i

0.0 ± 0.0g

2.33

0

2.69

37.5 ± 3.5de

0.0 ± 0.0f

0.0 ± 0.0c

1.6 ± 0.1d

0.3 ± 0.0c

1.3 ± 0.1a

2.5 ± 0.1bc

2.33

0

5.37

37.5 ± 17.7de

62.5 ± 17.7ab

0.0 ± 0.0c

1.8 ± 0.1c

0.8 ± 0.1a

0.9 ± 0.0c

2.7 ± 0.3b

2.33

0

10.74

25.0 ± 11.7ef

25.0 ± 11.7e

50.0 ± 0.0a

1.8 ± 0.0c

0.3 ± 0.0c

0.8 ± 0.0d

2.2 ± 0.1cd

4.65

0

2.69

62.5 ± 3.5bc

37.5 ± 3.5cde

0.0 ± 0.0c

2.0 ± 0.1a

0.3 ± 0.0c

0.6 ± 0.1e

2.0 ± 0.1de

4.65

0

5.37

50.0 ± 0.0cd

50.0 ± 0.0bcd

0.0 ± 0.0c

1.5 ± 0.0e

0.3 ± 0.0c

0.3 ± 0.0h

2.0 ± 0.1de

4.65

0

10.74

37.5 ± 5.9de

50.0 ± 0.0bcd

0.0 ± 0.0c

1.3 ± 0.0g

0.3 ± 0.0c

0.4 ± 0.0g

1.9 ± 0.1e

9.30

0

2.69

50.0 ± 0.0cd

25.0 ± 0.0e

25.0 ± 0.0b

1.4 ± 0.0f

0.3 ± 0.0c

0.4 ± 0.0g

1.5 ± 0.1f

9.30

0

5.37

16.7 ± 0.0fg

62.5 ± 17.7ab

0.0 ± 0.0c

1.4 ± 0.0f

0.3 ± 0.0c

0.6 ± 0.1e

1.3 ± 0.1f

9.30

0

10.74

70.8 ± 5.9b

29.2 ± 5.9de

0.0 ± 0.0c

1.6 ± 0.1d

0.5 ± 0.0b

1.0 ± 0.1b

1.9 ± 0.0e

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

When nodal segments were incubated with KIN and (2,4-D or NAA), results were statistically significant (P ≤ 0.05) in all variables, displaying a decrease in frequency of shoot induction (Table 5). However, shoot or callus were induced on 100.0 % of nodal segments using low concentrations of KIN (2.33 μM) along with NAA 2.69 μM or 5.37 μM 2,4-D, respectively. Furthermore, the average maximum simultaneous formation of callus and shoots (75.0 %) was also induced either using KIN 2.33 μM with NAA (5.37 or 10.74 μM), or KIN 9.30 μM with NAA 2.69 μM (Table 5), but few shoots per segment were formed (<3), whereas callus induced were small and browning. The highest mean number of shoots (3.0), mean number of nodes (2.4) and mean number of leaves per segment (2.9) were shown when nodal segments were incubated with 4.65 μM KIN and 2.69 μM NAA (Table 5). Some studies in Dioscorea genus have shown that BA is better than KIN in inducing a greater mean number of axillary shoots (Forsyth and Staden 1982; Poornima and Ravishankar 2007; Yan et al. 2011). Other studies have shown that the presence of BA adversely affects survival of explants (Martine and Cappadocia 1992) and may also decrease microtuber production (Islam et al. 2008). In our present study, however, we found that D. remotiflora displayed the best response in terms of percentages of shoot induction, mean number of shoots and mean number of nodes per segment using KIN alone or combined with NAA or 2,4-D in ranges from 2.33 to 9.30 μM. In this regard, induction response may differ depending to plant growth regulator type (Staba 1982).
Table 5

Effects of KIN and 2,4-D or NAA on shoots and/or callus induction of D. remotiflora Kunth nodal segments at 35 days of culture on MS medium

PGRs (μM)

Inductions (%)

Shoots/segment

Nodes/segment

Leaves/segment

Shoot length (cm)

KIN

2,4-D

NAA

Shoots

Callus

Shoots and callus

2.33

2.69

0

8.3 ± 11.8de

25.0 ± 0.0e

62.5 ± 17.7ab

2.6 ± 0.2b

0.5 ± 0.1e

1.9 ± 0.2cde

2.3 ± 0.1h

2.33

5.37

0

0.0 ± 0.0e

100.0 ± 0.0a

0.0 ± 0.0e

0.0 ± 0.0j

0.0 ± 0.0 g

0.0 ± 0.0j

0.0 ± 0.0k

2.33

10.74

0

0.0 ± 0.0e

50.0 ± 0.0cd

25.0 ± 0.0d

1.3 ± 0.0i

0.3 ± 0.0f

0.4 ± 0.0hi

5.1 ± 0.0a

4.65

2.69

0

29.2 ± 5.9c

45.8 ± 5.9d

25.0 ± 0.0d

1.5 ± 0.0h

0.3 ± 0.0f

0.6 ± 0.1h

2.8 ± 0.0fg

4.65

5.37

0

16.7 ± 11.8cd

12.5 ± 8.2ef

54.2 ± 8.2bc

1.8 ± 0.1fg

0.1 ± 0.0fg

0.3 ± 0.1ij

3.6 ± 0.1d

4.65

10.74

0

0.0 ± 0.0e

75.0 ± 11.7b

0.0 ± 0.0e

0.0 ± 0.0j

0.0 ± 0.0g

0.0 ± 0.0j

0.0 ± 0.0k

9.30

2.69

0

12.5 ± 5.9de

62.5 ± 5.9bc

25.0 ± 0.0d

2.0 ± 0.0def

0.0 ± 0.0g

1.0 ± 0.0g

2.0 ± 0.1i

9.30

5.37

0

0.0 ± 0.0e

100.0 ± 0.0a

0.0 ± 0.0e

0.0 ± 0.0j

0.0 ± 0.0g

0.0 ± 0.0j

0.0 ± 0.0k

9.30

10.74

0

12.5 ± 5.8de

75.0 ± 11.7b

0.0 ± 0.0e

1.3 ± 0.0i

0.0 ± 0.0g

0.4 ± 0.0 hi

1.5 ± 0.0j

2.33

0

2.69

100.0 ± 0.0a

00.0 ± 0.0f

0.0 ± 0.0e

2.4 ± 0.2c

1.1 ± 0.2bc

1.5 ± 0.0f

3.9 ± 0.1c

2.33

0

5.37

0.0 ± 0.0e

25.0 ± 11.7e

75.0 ± 11.7a

1.9 ± 0.0efg

1.3 ± 0.1b

2.1 ± 0.2bc

4.0 ± 0.1bc

2.33

0

10.74

12.5 ± 5.9de

0.0 ± 0.0f

75.0 ± 0.0a

1.6 ± 0.1gh

0.6 ± 0.1de

1.5 ± 0.1f

3.0 ± 0.1e

4.65

0

2.69

54.2 ± 7.0b

0.0 ± 0.0f

45.8 ± 7.0c

3.0 ± 0.2a

2.4 ± 0.2a

2.9 ± 0.1a

3.5 ± 0.1d

4.65

0

5.37

62.5 ± 5.9b

0.0 ± 0.0f

25.0 ± 0.0d

2.1 ± 0.2cde

0.8 ± 0.0d

2.3 ± 0.2b

2.3 ± 0.0h

4.65

0

10.74

16.7 ± 11.8cd

0.0 ± 0.0f

70.8 ± 5.9a

2.3 ± 0.1cd

1.0 ± 0.1c

1.8 ± 0.2def

2.6 ± 0.2 g

9.30

0

2.69

0.0 ± 0.0e

25.0 ± 11.7e

75.0 ± 11.7a

1.9 ± 0.0efg

1.1 ± 0.0bc

2.0 ± 0.1bcd

4.2 ± 0.2b

9.30

0

5.37

54.2 ± 5.9b

4.2 ± 0.0f

29.2 ± 5.9d

2.3 ± 0.1cd

1.0 ± 0.1c

1.6 ± 0.2ef

2.9 ± 0.1ef

9.30

0

10.74

62.5 ± 5.9b

0.0 ± 0.0f

25.0 ± 0.0d

2.6 ± 0.2b

0.6 ± 0.0de

1.9 ± 0.2cde

2.2 ± 0.1 h

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

Rooting of axillary shoots

Auxins are known for their ability to promote adventitious root formation, being IBA the most commonly used to obtain root initiation in conventional cutting (George et al. 2008). Effect of IBA showed significant differences (P ≤ 0.05) on nodal segments (Table 6). The maximum root induction (96.9 %) was shown in presence of 0.04 or 8.28 μM IBA, producing also a large average number of roots (5.9), mean number of shoots (2.2) and length of shoots (4.2 cm) with 8.28 μM IBA at 35 days of culture (Table 6, Fig. 1e). Similar studies have shown that addition of 4.9 or 9.8 μM IBA to the medium induced fastest rooting and had a higher average number of roots per segment in D. zingiberensis (Chen et al. 2003). The highest percentages of shoot induction (82.2–96.9 %) were produced using IBA ranging from 0.04 to 8.28 μM IBA, which were statistically equal. In addition, the greatest mean number of nodes (3.5) and mean number of leaves per segment (4.2) was expressed using 41.4 or 0.04 μM IBA, respectively. There are many factors known to influence the tuberization and rooting process, such as cytokinins and auxins (Chen et al. 2007). In fact, a study conducted by Mahesh et al. (2010) in D. wightii, found that the percentage of root induction decreases when adding cytokinins, whereas the number of abnormal axillary shoots increases by using high levels of these growth regulators. In contrast, addition of IBA has shown better effects on rooting percentage and number of roots per segment on D. oppositifolia and D. pentaphylla (Poornima and Ravishankar 2007).
Table 6

Effects of IBA on rooting of shoots and/or callus induction of D. remotiflora Kunth nodal segments at 35 days of culture on MS medium

PGRs (μM)

Induction (%)

Roots/segment

Shoots/segment

Nodes/segment

Leaves/segment

Shoot length (cm)

IBA

Roots

Shoots

Callus

0.04

83.8 ± 1.0b

87.5 ± 8.8a

0.0 ± 0.0c

3.2 ± 0.2b

2.3 ± 0.2a

2.7 ± 0.1b

4.2 ± 0.1a

3.6 ± 0.1b

0.41

96.9 ± 4.4a

93.8 ± 8.8a

0.0 ± 0.0c

2.8 ± 0.3b

2.1 ± 0.1a

2.1 ± 0.1 cd

4.0 ± 0.2ab

3.5 ± 0.3b

4.14

93.8 ± 8.8ab

96.9 ± 4.4a

0.0 ± 0.0c

3.5 ± 0.2b

1.8 ± 0.1b

1.8 ± 0.1de

3.6 ± 0.3ab

4.2 ± 0.3a

8.28

96.9 ± 4.4a

82.2 ± 1.5a

0.0 ± 0.0b

5.9 ± 0.4a

2.2 ± 0.2a

2.2 ± 0.2c

3.5 ± 0.2ab

4.2 ± 0.2a

20.70

71.2 ± 5.3c

41.5 ± 0.2b

13.9 ± 4.0b

5.3 ± 0.6a

1.7 ± 0.0b

1.4 ± 0.1e

2.9 ± 0.4c

3.1 ± 0.3b

41.40

88.5 ± 1.5ab

39.6 ± 4.7b

23.9 ± 0.3a

3.1 ± 0.3b

1.4 ± 0.0c

3.5 ± 0.3a

1.9 ± 0.1d

1.5 ± 0.1c

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

Since plants have a high composition of water and this amount depends of their environment (which is very difficult to control), using dry biomass as other measure of plant growth tends to be more reliable. Therefore, mean number of tuberous roots per segment and mean weights of dry biomass in vitro (shoots, roots or tuberous roots) were also examined. Results displayed significant differences (P ≤ 0.05) by using different concentrations of IBA (Table 7). In fact, the highest mean weight of shoots per segment (79.8 mg DW), mean weight of roots per segment (15.1 mg DW) and mean number of tuberous roots per segment (6.0) were obtained with 8.28 μM IBA at 90 days of culture (Table 7, Fig. 1f). Whilst, an increasing on the average dry weight of tuberous roots (54.3 mg DW) and mean dry weight per tuberous root (36.5 mg DW) was produced adding to culture medium 20.70 μM IBA, showing that, by increasing the mean number of tuberous roots per segment, the individual weight of them is decreased. In this regard, it was found that BA inhibits microtuber induction. By contrast, IBA has shown striking promotive effects on microtuber induction and growth. IBA promoted also grater effects than cytokinins on in vitro shoots growth in D. composita (Alizadeh et al. 1998).
Table 7

Effect of IBA on biomass and number of tuberous roots of D. remotiflora Kunth axillary shoots at 90 days of culture on MS medium

PGR (μM)

Biomass/segments (mg DW)

Biomass/tuberous root (mg DW)

Tuberous roots/segment

IBA

Shoots

Roots

Tuberous roots

0.04

18.4 ± 0.4d

2.6 ± 0.1c

3.6 ± 0.2d

3.2 ± 0.7de

1.2 ± 0.2cd

0.41

15.3 ± 1.1d

2.6 ± 0.1c

0.8 ± 0.3e

1.2 ± 0.4e

0.7 ± 0.0d

4.14

66.4 ± 1.1bc

9.7 ± 2.9b

51.8 ± 1.4b

13.4 ± 3.5bc

4.0 ± 0.9b

8.28

79.8 ± 0.9a

15.1 ± 0.6a

48.9 ± 0.2b

8.3 ± 1.3cd

6.0 ± 0.9a

20.7

69.0 ± 0.9b

7.3 ± 1.6b

54.3 ± 2.4a

36.5 ± 4.1a

1.5 ± 0.2cd

41.4

63.4 ± 3.0c

8.4 ± 1.3b

36.5 ± 0.1c

17.0 ± 1.8b

2.2 ± 0.2d

Data represent mean ± SE

Values with different superscript letters in the same column are significantly different according Duncan’s multiple-range test (P ≤ 0.05)

Conclusions

The present study has shown the promotive effects of exogenous PGRs on axillary shoots induction and callus formation on D. remotiflora in vitro culture. Kinetin was the cytokinin most suitable for shoot formation, number of shoots, number of nodes, number of leaves and shoot length, using low concentrations (2.33 μM) on MS culture medium. Combinations of KIN with any auxins affected proliferation of axillary shoots negatively. Exogenous addition of NAA induced callus and/or shoots formation; whereas, AIB was essential to roots induction and tuberous roots formation in all concentrations used. A regeneration system for D. remotiflora Kunth by inducing shoots and tuberous roots in vitro was done. These results can be used as a protocol for an extensive system of cultivation to produce tubers and for optimizing in vitro conditions for callus production.

Acknowledgments

Research reported here was carried out during a postdoctoral stay supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) (project 99963) and the Consejo Estatal de Ciencia y Tecnología del Estado de Jalisco (COECYTJAL) (project PS-2009-919).

Copyright information

© Springer Science+Business Media B.V. 2012