A combination of vermiculite and paper pulp supporting material
for the photoautotrophic micropropagation of sweet potato
F. Afreen-Zobayed
a,*, S.M.A. Zobayed
a, C. Kubota
a, T. Kozai
a, O. Hasegawa
baLaboratory of En6ironmental Control Engineering,Faculty of Horticulture,Chiba Uni6ersity,Matsudo,Chiba 271-8510, Japan bNisshinbo Industries Inc. 1-18-1 Nishiarai Sakaecho,Adachi-ku,Tokyo 123, Japan
Received 14 January 2000; received in revised form 1 May 2000; accepted 2 May 2000
Abstract
A mixture of vermiculite (hydrous silicates) and paper pulp (waste product of paper industry) was used as a supporting material for the in vitro photoautotrophic micropropagation of plantlets. Sweet potato was used as a model plant to find out the appropriate proportion of vermiculite and paper pulp for the optimum growth of the plantlets. The plantlets grown in the conventional supporting material, agar, were used as the control. The study revealed that in all aspects, the plantlets grown in vermiculite mixed with 30% (w/w) paper pulp exhibited the highest growth performance. The shoot and root fresh mass were
×2.7 greater than those in agar (control); the leaf, stem and root dry mass were also greater and at least two fold in this treatment compared with those in the control. The net photosynthetic rate per plantlet was highest in this treatment, and on day 20 it was 15.3mmol CO2h−1as compared with 9.8mmol CO2h−1in the control. The growth of both shoots and roots decreased gradually with the increase or decrease of percentage of paper pulp in the supporting material. In general, the growth was significantly poorer in the plantlets grown in 100% vermiculite than that in vermiculite mixed with 30% paper pulp but still greater than in the control. The porosity of the supporting materials increased with the increase in the percentage of paper pulp in the supporting material. After transplanting to the ex vitro condition the survival percentage did not vary significantly (90 – 100%) among the treatments, except in control where it was only 73%. The number of unfolded leaves and the stem height were similar among the treatments except those in the control. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Agar; Ex vitro; Florialite; In vitro; Porosity; Survival
www.elsevier.com/locate/plantsci
1. Introduction
The nature of the supporting material for root-ing exerts considerable influence on the growth and quality of plantlets in vitro. Since root and shoot development are inter-dependent, poor root development will eventually affect the shoot devel-opment. Agar, the conventional gelling agent, has a number of drawbacks such as lowering the oxy-gen diffusion coefficient [1], affecting the differen-tiation and development of cultured tissues, negatively in many cases [2], limited adaptability
to automation, inability to circulate the nutrient medium in the culture vessel, difficult to clean the roots and the vessel before transplanting and sig-nificant difference in the sensitivity of the plant species to different agar brands [3].
Use of fibrous materials such as cellulose [4], rock wool [5] and artificial soil such as vermiculite [4,6] have been reported to enhance the growth of plants in vitro. In our previous study we demon-strated that the use of Florialite (Florialite; Nissh-inbo Industries, Inc. Tokyo) had a significant stimulatory effect on the growth of in vitro sweet potato plantlets [7]. The supporting material, Flo-rialite, is a mixture of vermiculite and paper pulp, and is able to absorb at least 71% nutrient solu-tion of its own volume. The growth of the plantlets obtained in Florialite was greater than
* Corresponding author. Present address: Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK. Fax:+ 44-1482-465458.
E-mail address:afreen@green.h.chiba-u.ac.jp (F. Afreen-Zobayed).
that of the vermiculite indicating that the addition of paper pulp with vermiculite in the supporting material was the major reason for this greater growth. Moreover, the solid cubes of the Florialite makes it possible to automate micropropagation and handling of material more easily and precisely compared with agar or vermiculite granules. Infor-mation, as the proportion of the components and the effect of Florialite on the growth of the plants after transplanting ex vitro is unavailable. There-fore, sweet potato cuttings were grown photoau-totrophically in vitro in supporting material which was either 100% vermiculite or vermiculite mixed with 90, 70, 50, 30, 17 and 10% (w/w) paper pulp. The paper pulp used in the supporting materials is the waste product of a paper industry (Nisshinbo Industries, Inc. Tokyo). The effects of different supporting materials on the growth of the sweet potato plantlets after transplanting ex vitro were also examined.
2. Materials and methods
2.1. Plant material, treatments and culture condition
Single nodal cuttings (fresh mass=116 mg)
each with a leaf of photomixotrophically grown sweet potato (Ipomoea batatasL. (Lam.), cv. Beni-azuma) plantlets were cultured (four explants per Magenta-type vessel; volume 385 ml). MS [8] was used (45 ml per vessel) as a basal medium and vitamins and sucrose were excluded from the for-mulation to ensure photoautotrophic conditions. The supporting materials (45 ml per vessel) were vermiculite 100% and vermiculite mixed with 10, 17, 30, 50, 70 or 90% (w/w) paper pulp. Agar (8 g l−1; Kanto Chemical Co., Inc. Japan) was used as the control. Each treatment consists of six repli-cates. To provide natural ventilation, three gas permeable filter membranes (pore diameter, 0.5 mm; Millipore, Japan) were attached covering the holes (diameter 10 mm) of the lid of each of the vessels. The number of air exchanges of the vessel was 4.4 h−1, estimated according to Kozai et al. [9] throughout the culture period.
Culture vessels were placed at 2791°C air tem-perature during photoperiod and under cool-white fluorescent lamps and 16 h photoperiod and 8 h dark period in the growth room. The CO2
concen-tration of the growth room during the photope-riod was maintained at ca. 900 – 1000 mmol mol−1 and the photosynthetic photon flux (PPF) was 150 mmol m−2 s−1 (measured on the empty culture shelf). The relative humidity in the growth room was maintained between 75 – 80%. To avoid possi-ble damage of the explants especially by water stress during transferring cuttings, vessels were
placed under 70 mmol m−2 s−1 (PPF) without
ventilation (with all the filters closed) for the first 48 h.
Plantlets were harvested on day 21 and the growth was studied in terms of leaf number and area, fresh and dry mass of leaves, roots and stems. After 21 days of culture, plantlets (15 plantlets from each treatment) were transplanted in the plastic pots (80 mm diameter) with a mix-ture of compost and vermiculite (1:1 by volume). Plants were then placed in a greenhouse (Septem-ber 17 – Octo(Septem-ber 22, 1998; temperature 2992°C; RH 60 – 70%) and the percentage survival was recorded on day 9 after transplanting to the green-house. The stem height and number of unfolded leaves were recorded on days 9, 18 and 30.
2.2. Measurement of CO2 concentration, net
photosynthetic rate, percentage porosity and statistical analysis
The CO2concentration inside the culture vessels was determined under steady state conditions dur-ing the photoperiod on days 0, 7, 14, 17 and 20 by removing 250 ml samples of gas from the culture vessels for analysis by gas chromatography (GC-12A, Shimadzu Co., Ltd., Kyoto, Japan).
Net photosynthetic rate per plantlet, Pn (mol
h−1/plantlet) was calculated after the method of Fujiwara et al. [10] by using the following equation:
Pn={k*E*V*(Cout−Cin)}/N
k is the conversion factor of CO2from volume to mole; E, the number of air exchanges per hour of the culture vessel (h−1); V, the air volume of the culture vessel (l); Cin and Cout, CO2concentrations (mol mol−1) inside and outside the culture vessel under steady state conditions during photoperiod;
N, the number of plantlets per vessel.
material was kept in the oven (60°C) for 48 h and then placed in a specially designed acrylic box in which the material fits exactly. Water was added gradually until the supporting material became fully saturated. Percentage porosity (P) was then calculated as following:
P=(W/S)*100
P is the percentage porosity of the substrate; W, volume of added water; S, total substrate volume (volume of the dry substrate+volume of the air inside the substrate).
Statistical significance was determined by one-way analysis of variance (ANOVA) and Duncan’s Multiple Range Test. The experiment was con-ducted twice.
3. Results and discussion
3.1. Growth and de6elopment
The use of vermiculite with 30% paper pulp was found to stimulate growth of leaves, stems and roots significantly, and any further increase or decrease in the proportion of paper pulp brought about a gradual reduction in the shoot and root growth (Table 1; Fig. 1).
After 21 days of culture, the leaf fresh and dry mass and leaf area of the plantlets grown on 30%
paper pulp were ×2.7, ×2.8 and ×2,
respec-tively, those of the control. The most significant development was observed in the root system of plantlets grown on 30% paper pulp where root dry
mass was ×3.8 that of the control; the stem fresh
and dry mass were also higher (×2.6) in this
treatment compared with those of the control. The results accord with the previous findings of Ichimura and co-workers who observed that the pulp extract stimulated root growth of several vegetables [11] and stimulated shoot regeneration from the explants of tomato [12].
Taking into account the growth of both shoot and roots the second greatest performance was by plantlets grown on either 17 or 50% paper pulp followed by 70% paper pulp, except the number of leaves was greatest for plantlets grown on 70% paper pulp. The difference in growth was less noticeable between 10 and 90% paper pulp treat-ments. The growth (shoot and root) decreased further with 100% vermiculite. In our previous work [7] agar had a depressing effect on the growth of the plantlets apart from the number of leaves and this was confirmed in this study.
The clearest difference that emerged between the mixture of vermiculite and paper pulp treatments and the control was in the development of the root system. The main adventitious root gave rise to fine laterals for those plantlets grown in the mix-ture of vermiculite and paper pulp (Fig. 1). The number of laterals was highest in the 30% paper pulp treatment and decreased gradually with any increase or decrease in the proportion of paper pulp (data not shown). In the control the main adventitious root produced sparse, short laterals that might be related to the poor shoot growth of the plantlets. The presence of lateral roots could have resulted in a higher nutrient absorption
ca-Table 1
Effects of different proportions of vermiculite and paper pulp on growth of 21 day old in vitro-grown sweet potato plantletsx,y
Stem Root
xMeans within a column followed by different letters are significantly different atP50.05 by Duncan’s Multiple Range Test.
Fig. 1. Root growth of sweet potato plantlets cultured photoautotrophically for 21 days in supporting material consisting of various percentages of vermiculite (v): paper pulp (p). (a) 100% vermiculite; (b) vermiculite: paper pulp (V:P) 90:10; (c) V:P 83:17; (d) V:P 70:30; (e) V:P 50:50; (f) V:P 30:70; (g) V:P 10:90; (h) agar.
pacity, as the laterals are more permeable than the main adventitious root [13]. It was interesting to note that in a mixture of vermiculite and paper pulp, the fibres of the paper pulp form a net like structure with vermiculite granules randomly ar-ranged in it (Fig. 2). The compact nature of this material probably facilitates the formation of lateral.
Earlier reports suggested that the use of cellu-lose plugs (Sorbarod Baumgartner Rapiers SA, Switzerland) improved the growth of in vitro grown plantlets [4]. In our study, among the paper pulp treatments although the lowest leaf, stem and root fresh and dry mass (983, 126, 673, and 83, 10, 61 mg respectively) were observed in 10% paper pulp treatment, however, these values were ×1.9,
×1.6, ×2.5 and ×2, ×1.5 and ×3.3,
respec-tively than those of the agar. It was concluded that the root stimulating activity resulted from the presence of paper pulp in the supporting material. For many years paper mill sludge has been used as a rooting substrate in forestry [14], to prepare woody perennial nursery stocks [15], to grow
or-namental plants in nursery container [16,17], in agriculture [18,19] and also, for deciduous land-scape shrub cuttings [20]. Ichimura and Oda [11] showed that pulp extract contains three kinds of root growth-stimulating substances that are highly polar compounds of low molecular weight.
3.2. Net photosynthetic rate
It was not surprising that the net photosynthetic rate per plantlet followed a similar trend to the growth of the plantlets (Fig. 3), and the highest rate of net photosynthesis (15.3 mmol CO2 h−1 plantlet on day 20) was ×1.5 that of plantlets grown in agar. The photosynthetic rates for the other treatments decreased in accordance with the decrease or increase of paper pulp ratio in the supporting material in which they had been grown.
among 50, 70 and 10% paper pulp treatments was not significant. The lowest net photosynthetic rate (9mmol CO2h−
1/plantlet) on day 20 was recorded
Fig. 4. (a) Percentage porosity of different supporting materi-als; (b) percentage survival ex vitro of sweet potato plants previously grown photoautotrophically (PPF was 150 mmol
m−2 s−1) for 21 days in supporting material consisting of various percentages of vermiculite (v): paper pulp (p). Error bars represent 9SE.
Fig. 2. Supporting material consisting of vermiculite and paper pulp (X12).
Fig. 3. Net photosynthetic rates of sweet potato plantlets grown photoautotrophically (PPF was 150 mmol m−2 s−1)
for 21 days in supporting material consisting of various percentages of vermiculite (v): paper pulp (p). Error bars represent 9SE.
in agar-grown plantlets as expected since the growth of the plantlets was poor (Table 1). The results are in close agreement with our previous findings [7]. In another study Kirdmanee et al. [4] confirmed that the net photosynthetic rate of Eu
-calyptus was higher when grown in 100% vermi-culite compared with that of other gelling agents.
3.3. Porosity of the supporting material
porosity was nearly 73%. In agar, the porosity should be nearly zero after gelling.
3.4. Sur6i6al percentage and growth after transplanting
The ex vitro growth is a closer reflection of the in vitro development of the plantlets, and in ex vitro the differences in growth of the plants in
fibrous materials and agar became more apparent. There was no significant difference in the percent survival among the treatments except in the con-trol (agar) where 73% plants survived after trans-planting (Fig. 4b), and the leaf and stem growth was slower compared with the plants of the other treatments. Almost 98 – 100% plants survived in the 30, 17 and 50% paper pulp treatments fol-lowed by 95% survival in the 70% paper pulp and 92 – 93% survival in the 10 and 90% paper pulp and 90% survival in 100% vermiculite treatments. Some leaves became permanently wilted in the survived plants (data not shown).
The stem height of the plants slightly decreased in accordance with the growth of the plantlets in vitro for the different treatments in which they had been grown previously (Fig. 5a). The number of unfolded leaves among the treatments was simi-lar except in agar where the number was slightly smaller (9.4 on day 30) compared with that of 30% paper pulp treatment (11.4) (Fig. 5b) this accords with the smaller stem height (×0.6 that of 30% paper pulp) of the plants.
4. Conclusions
The results presented in this study represented a clearer understanding of the more detailed aspects of the supporting material used for the microprop-agation of sweet potato. To summarise, vermi-culite mixed with 30% paper pulp used as the supporting material enhanced root and shoot growth (×3.8 and B×2, respectively that of the control) of the plantlets in vitro and enabled 98 – 100% of the plants to acclimatise successfully ex vitro. In contrast, the poorly developed plantlets in agar showed the lowest percentage (73%) sur-vival ex vitro, and growth of the survived plants was slow. It was also revealed that the use of waste product of the paper industry in the mi-cropropagation technique is an environmentally sound alternative for disposing or reusing these wastes.
Acknowledgements
We are grateful to Mr Yohjiroh Ohno of Nissh-inbo Industries Inc., Japan, for kindly supplying the materials. The financial support from the
Fig. 5. (a) Stem height and (b) number of unfolded leaves ex vitro of sweet potato plants previously grown photoau-totrophically (PPF was 150 mmol m−2 s−1) for 21 days in
Japanese Society for the Promotion of Science (JSPS) Research-for-the-Future Program
(Pro-ject no. JSPS-RFTF 96L00605) is greatly
acknowledged.
References
[1] K. Fujiwara, C. Yamauchi, T. Kozai, Effects of culture media components on the oxygen diffusion coefficient in liquid and gelled media, Abstr. Japan. Plant Cell Tissue Cult. Ann. Meeting, Kyoto. (1993) 40.
[2] K. Ichimura, M. Oda, Stimulation of root growth of several vegetables by extracts from a commercial prepa-ration of agar, J. Jap. Soc. Hort. Sci. 67 (Suppl. 3) (1998) 341 – 346.
[3] H.J. Scholten, R.L.M. Pierik, Agar as a gelling agent: differential biological effects in vitro, Sci. Hort. 77 (1 – 2) (1998) 109 – 116.
[4] C. Kirdmanee, Y. Kitaya, T. Kozai, Effects of CO2 enrichment and supporting material in vitro on photoau-totrophic growth of Eucalyptus plantlets in vitro and ex vitro, In Vitro Cell. Dev. Biol.-Plant. 31 (1995) 144 – 149. [5] T. Kozai, Acclimatization of micropropagated plants, in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, High tech and Micropropagation I, vol. 17, Springer-Verlag, Berlin Heidelberg, 1991, pp. 127 – 141. [6] C. Kirdmanee, Y. Kitaya, T. Kozai, Effects of CO2
enrichment and supporting material in vitro on photoau-totrophic growth of Eucalyptus plantlets in vitro and ex vitro: Anatomical comparisons, Acta Hort. 393 (1995) 111 – 115.
[7] F. Afreen-Zobayed, S.M.A. Zobayed, C. Kubota, T. Kozai, O. Hasegawa, Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically, In Vitro Cell. Dev. Biol.-Plant. 35 (1999) 470 – 474.
[8] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Phys-iol. Plant. 15 (1962) 473 – 497.
[9] T. Kozai, K. Fujiwara, I. Watanabe, Fundamental stud-ies on environments in plant tissue culture vessels. (2) Effects of stoppers and vessels on gas exchange rates
between inside and outside of vessels closed with stop-pers, J. Agr. Met. 42 (2) (1986) 119 – 127 (Japanese with English abstract).
[10] K. Fujiwara, T. Kozai, I. Watanabe, Fundamental stud-ies on environments in plant tissue culture vessels. (3) Measurement of carbon dioxide gas concentration in closed vessels containing tissue cultured plantlets and estimates of net photosynthetic rates of plantlets, J. Agr. Met. 43 (1987) 21 – 30 (Japanese with English abstract). [11] K. Ichimura, M. Oda, Stimulation of root growth in several vegetables by wood pulp extract, J. Japan Soc. Hort. Sci. 63 (Suppl. 4) (1995) 797 – 803.
[12] K. Ichimura, T. Uchiumi, K. Tsuji, M. Nagaoka, Effects of medium supports on the growth of adventitious buds from cotyledons of tomato (Lycopersicon esculentum), J. Japan Soc. Hort. Sci. 59 (Suppl. 2) (1990) 344 – 345 (In Japanese).
[13] W. Armstrong, Aeration in higher plants, in: H.W. Woolhouse (Ed.), Advances in Botanical Research, vol. 7, Academic Press, London, 1979, pp. 229 – 289. [14] D.G. Brockway, Forest floor, soil, and vegetation
re-sponses to sludge fertilization in red and white pine plantations, J. Soil Sci. Soc. Am. 47 (1983) 776 – 784. [15] C. Chong, L. Daigneault, Influence of IBA
concentra-tions on rooting of woody perennial nursery stock, Comb. Proc. Intl. Plant. Prop. Soc. 36 (1986) 108 – 115. [16] C. Chong, R.A. Cline, Response of four ornamental shrubs to container substrate amended with two sources of raw paper mill sludge, HortScience 28 (1993) 807 – 809.
[17] K.L. Bellamy, C. Chong, R.A. Cline, Paper sludge uti-lization in agriculture and container nursery culture, J. Environ. Qual. 24 (1995) 1074 – 1082.
[18] Bellamy, K.L., deLint, N., Pridham. N.F., Cline, R.A., Agricultural utilization of paper mill sludge in the Nia-gara area, In. Proc. 13th Intl. Symp. Wastewater Treat-ment and 2nd Workshop on Drinking Water, Montreal, QC, 1990, pp. 65 – 81.
[19] M.N. Aitken, J.G. Lewis, B. Evans, Effects on soil fertility from applying paper mill sludge to agricultural land, Soil Use Manage. 11 (1995) 152 – 153.
[20] C. Chong, B. Hamersma, K.L. Bellamy, Comparative rooting of deciduous landscape shrub cuttings in media amended with paper mill biosolids from four different sources, Can. J. Plant. Sci. (1998) 519 – 526.
