G.R. Rout, S.K. Palai and P. Das

Plant Biotechnology Division, Plant Tissue Culture Laboratory, Regional Plant Resource Centre, Bhubaneswar- 751 015, India. Fax: 0091-674-550274. E-mail: grrout@hotmail.com

Received 07-X-2000. Corrected 10-V-2001. Accepted 28-V-2001.

The induction of rooting in microshoots of Zingiber officinale cvs. Suprava, Turia local, Suruchi and V₃S₁₈ was achieved on half-strength basal Murashige and Skoog's medium supplernented with 0.5-1.0 mg/l either indole-3-acetic acid (lAA) or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose within 7-9 days of culture. Rooting was inhibited when the microshoots were cultured under higher concentration of auxins. The microshoots cultured on medium supplemented with NAA induced large number of thin root hairs with friable calluses within 6-7 days. Peroxidase activity was deterrnined during root induction (0-day to the 10^th day at every 2 day interval) from microshoots derived in vitro. The activity was minimum in the inductive phase (primary) and at the maximum level during the root initiative phase. These finding may be useful in monitoring the rooting behaviour in microshoots derived from different subculture and peroxidase activity as a marker for root initiation.

Key words: In Vitro, medicinal plant, peroxidase activity, root initiation. shoot multiplication.
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Ginger, Zingiber officinale Rosc. (Zingiberaceae), is an important, tropical, horticultural plant valued in culinary and pharmaceutical preparations. Cultivation of the plant is limited by diseases spread through vegetative propagation and limiting supply of high quality rhizomes. The quality of the rhizome depends on the age and a the harvesting time. The oil content in plantlets derived from in vitro cultures could also be controlled by the composítion of the culture medium. High-quality volatile oils and higher rhizomes yield in ginger would be of great benefit to pharmaceutical industries. ln vitro culture techniques provide an alternative way of plant propagation and function as a tool for crop improvement programs (Vasil 1988). Rootíng of microshoots is critical in plant production systems in vitro. Induction of rooting depends on a series of interdependent phases (induction, initiation and expression) (Gaspar et al., 1994). Various studies on adventitious root formation have shown the fundamental role played by peroxidase in rooting of plants cultured in vitro (Berthon et al. 1989, Rival et al. 1997). The effect of auxins with regard to the peroxidase activity in rooting of various plant species has also been reported by Kevers et al. (1997). The aim of the present investigation is to describe the effect of auxins on rooting and analyse the peroxidase activity during root induction in microshoots of Z. officinale cvs. Suprava, Turia local, Suruchi and V₃S₁₈.

Materials and methods

Plant material: Mature rhizomes of Z. officinale cvs. Suprava, Turia local, Suruchi and V₃S₁₈ were collected at the High Altitude Research Station of the Orissa University of Agriculture and Technology, Pattangi, Orissa, India and incubated in a bed of sterile, moist, sandy soil (65 % relative humidity with 26+2 °C) to induce sprouting. The pale-white sprouting buds (after 30 days of incubation) were collected and washed with 2 % (v/v) detergent solution 'Teepol' (Qualigen, India) for 15 min, subsequently surface sterilised using 0.1 % (w/v) aqueous solution of mercuric chloride for 25 min followed by several rinses in sterile distilled water. The shoot bods were dissected under a dissecting microscope; the meristems with apical dome or with one or two leaf primordia ranging in size from 0.1-0.5 mm were removed and used as explants.

Culture medium: Isolated shoot meristems were placed on Murashige and Skoog (1962) (MS) basal salts supplemented with various concentrations of cytokinins, i.e. 6-benzylaminopurine (BA: 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0 and 8.0 mg/l), kinetin (Kn: 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0 and 8.0 mg/l), and adenine sulfate (Ads: 50, 100, 150 and 200 mg/l), and auxins like indole-3acetic acid (IAA: 0.0, 0.5, 1.0, 1.5 and 2.0 mg/l), indole-3-butyric acid (IBA: 0.0, 0.5, 1.0, 1.5 and 2.0 mg/l) and 1-naphthaleneacetic acid (NAA: 0.0, 0.5, 1.0, 1.5 and 2.0 mg/l) for shoot multiplication. The pH of the medium was adjusted to 5.8 prior to autoclaving. All cultures were incubated under a 16 hr photoperiod having a light intensity of 55 µmol m^-2s^-1 from cool, white fluorescent lamps at 25 + 2 °C.

Induction of rooting and transplantation: Microshoots (1-2 cm) were separated from the initial culture as well as different subcultures and transferred into various semi-solid basal half-strength MS medium alone or in combination with IAA, IBA or NAA (0.0, 0.25, 0.50, 1.0 and 1.5 mg/l) and 2 % (w/v) sucrose for root induction. In another set of experiments, the microshoots derived from the multiplication medium were cultured for 7 days on basal MS medium and subsequently transferred to the rooting medium (1/2 MS + 0.5-1.0 mg/l IAA or IBA + 2 % (w/v) sucrose) for induction of rooting. The incubation conditions were the same as mentioned earlier for shoot growth. The rooted plantlets were removed from the culture vessels, washed gently under running tap water and planted in earthen pots containing a mixture of soil and sand (3: 1) (v/v). The plantlets were acclimatized for one month in the greenhouse (85 % relative humidity at 30 + 2 °C).

Sample collection: Microshoots were collected from the mother cultures prior to inoculation into the rooting medium (0-day) and at every two day intervals up to 10 days. The experiment was repeated three times.

Scoring of data and statistical analysis: All the cultures were examined periodically. The initiation of root from microshoots was recorded on the basis of visual observations. There were 20 replicates per each treatment and the experiment was repeated at least three times. Subculturing of shoot multiplication was carried out at an interval of four weeks. The effects of different treatments were quantified on the basis of percentage of culture showing response for multiplication, rooting and the number of roots/shoot. The data were analysed statistically by the Duncan's Multiple Range test (Harter 1960).

Results

Shoot multiplication: Multiplication of shoot was achieved from apical meristems of Z. officinale cvs. Suprava, Turia local, Suruchi and V₃S₁₈ on MS basal medium containing 4.0-6.0 mg/l BA supplemented with 1.0-1.5 mg/l IAA and 150 mg/l adenine sulfate. The rate of shoot multiplication of different cuItures reached a maximum value on medium having 6.0 mg/l BA, 1.5 mg/l IAA and 150 mg/l adenine sulfate (Fig. 1a). Of the three auxins tested, IAA was the most effective for shoot proliferation (data not shown). There was no sign of shoot multiplication when explants were cultured in media devoid of cytokinin. At lower concentrations of BA, the rate of shoot proliferation decIined. The rate of shoot multiplication was stable and constant up to one year without decIination. ]]>

Fig. 1. a. Induction of multiple shoots from shoot meristems of Zingiber
officinale cv. Turia local on MS medium supplemented with 6.0 mg/l BA,
1.5 mg/l IAA and 150 mg/l adenine sulfate after 4 weeks of culture (Bar = 20 mm).
 b. Induction of rooting from microshoots of Zingiber officinale cv. Turia local on 1 / 2
strength MS semisolid medium supplemented with 1.0 mg/l IAA after 10 days of ]]> culture (Bar = 10 mm).

Induction of rooting from microshoots: Elongated microshoots (1-2 cm) were rooted on half-strength basal MS medium supplemented with various concentrations of either IAA or IBA depending on the cuItures tested (Table 1). The rooting in the microshoots was inhibited in the medium devoid of growth regulator. Though NAA induced roots at lower concentration, callusing appeared at the basal cut end. Root initials were observed within 7-8 days of culture which developed into a good root system within 8-10 days of cuIture (Fig. 1b). Rooting ability was slowed on the medium containing higher concentrations of IAA or IBA (1.0-1.5 mg/l). However, optimal, rooting in 'Suprava', 'Turia local', 'Suruchi' and the shoots developed root hairs with intervening calluses at higher concentrations. The rooting ability was decreased when microshoots derived from the 4^th subculture. The microshoots grown on a cytokinin rich medium and subsequently transferred to rooting medium did not give any significant response for induction of rooting. The microshoots grown on a hormone free medium for 7 days and subsequently transferred to rooting medium showed positive response.

Peroxidase activity: The peroxidase activity in microshoots subjected to different treatments during the rooting process was determined (Table 2). The activity was at the minimum in the primary (inductive) phase and at the maximum in the secondary (initiative) phase in the microshoots grown on medium having 0.25-1.0 mg/l IAA or IBA (Table 2). The minimum activity was noted between the 0 and 2^nd day; the maximum activity, however, was noted from the 6^th to the 8^th day of culture.

15 replicates/treatment; repeated thrice

Acclimatization and field establishment: About 95 % of the rooted plantlets established in the greenhouse within two-three weeks of transfer. The plants grew normally and attained six-eigth cm within 30 days of transfer.

Discussion

The present study showed that it is possible to induce multiple shoots and rooting from apical meristems of Z. officinale cvs. Suprava, Turia local, Suruchi and V ₃S₁₈ on medium containing different concentrations of cytokinin and auxin. The combination of auxin and cytokinin influencing the production of shoots and the subsequent elongation of some ginger cultures was well documented (Malamug et al. 1991). The multiple shoots were rooted on half-strength MS semisolid medium supplemented with 0.5-1.0 mg/l IAA or IBA within 7-8 days of culture. The percentage of microshoots forming roots and the number of roots/shoot varied significantly with different concentrations of IBA or IAA; the shoots developed roots with intervening calluses at higher concentrations. The application of exogenous auxins promoting rooting in different plant species was reported (Blazkova et al. 1997, Kevers et al. 1997). The rooting ability decreased when microshoots derived from the 4^th subculture. There are few reports on the effect of age of microshoots on rooting behavior (DeKlerk et al. 1997). Enzymes are known as metabolic markers which change during development and differentiation (Scandalios 1969). It is clear that auxin treatments induced a sharp increase in the peroxidase activity during the course of the experiment and this might be due to absorption of auxin during the multiplication stage. Some researchers suggest that auxin enters the cut surfaces and it is rapidly taken up in the cell wall by pH trapping (Rubery and Sheldrake 1973) and by influx carriers (Delbarre et al. 1996). Epstein and Ludwig-Muller (1993) reported that during the course of root induction, there are two major pathways of conversion: oxidation and conjugation. Exogenous auxins applied to microcuttings became oxidised, resembling the enzymes involved in the wounding reactions as reported by DeKlerk et al. (1999). They also indicated that !AA or IBA oxidation caused by non-specific peroxidases is related to the wounding response. The peroxidase activity was also at a minimum in the primary (inductive) phase and at the maximum level in the secondary (initiative) phase in the microshoots grown on medium having 0.5-1.0 mg/l IAA or IBA. The minimum activity was noted between day 0 and 2; the maximum activity, however, was noted from the 6^th-8^th day. The pattern of enzyme activity was similar as reported earlier in other plant systems (Berthon et al. 1990, Rival et al. 1997). Changes in the levels of peroxidase activity in relation to auxin treatment have been reported in other plants (Moncousin et al. 1988, Gaspar et al. 1994).

The present study has confirmed the role of auxins on induction of rooting ability of Z. officinale cvs. Suprava, Turia local, Suruchi and V₃S₁₈ and showed the increased level of peroxidase activity at different intervals during the process of rhizogenesis. Various studies on other plant species (Asparagus, Populus and Prunus) have shown the fundamental role played by peroxidase activity during root initiation and expression (Hausman 1993). The IAA or IBA oxidation is caused by peroxidases related to the wounding response as reported by DeKlerk et al. (1999). This study may be useful in monitoring the rooting beahaviour in microshoots and to characterize the molecular events during rhizogenesis.

The authors wish to acknowledge to the Department of Forest and Environment, Govemment of Orissa for necessary facilities.

La inducción de enraizamiento en microtallos de Zingiber officinale cvs. Suprava, Turia local, Suruchi y V₃S₁₈ fue lograda en medio de Murashige y Skoog basal de dureza media suplementado con 0.5-1.0 mg/l ya sea con ácido 3-indol acético (lAA) o ácido 3-indol butírico (IBA) y 2 % (w/v) de sucrosa dentro de 7-9 días de cultivo. El enraizamiento fue inhibido cuando los microtallos fueron cultivados bajo concentraciones más altas de auxinas. Los microtallos cultivados en un medio suplementado con NAA indujeron un gran número de pelos de raíz con callos frágiles dentro de 6-7 días. La actividad de la peroxidasa fue determinada durante la inducción de enraizamiento (de 0 a 10 días, de cada 2 días) de microtallos derivados in vitro. La actividad fue mínima en inductiva (primaria) y máxima en la fase inicial Estos resultados pueden ser útiles para monitorear, el comportamiento de enraizamiento en microtallos derivados de diferentes subcultivos y la actividad de la peroxidasa como un marcador de inicio de enraizamiento.