Environmentally safe in vitro regeneration protocol for Curcuma, Kaempferia and Zingiber

1 Gene Research Center, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. 2 Institute of Biotechnology and Genetic Engineering (IBGE), NWFP Agricultural University Peshawar-Pakistan. 3 Plant Breeding Laboratory, Faculty of Agriculture, Padjadjaran University, Indonesia. 4 Plant Genetic Resources Program, National Agricultural Research Center, Islamabad45500, Pakistan.


INTRODUCTION
The sustainable utilization and conservation of plant genetic resources is assisted and accelerated if collections are correctly evaluated, characterized and properly maintained (Hussain et al., 2008).Due to the fast occurring genetic erosion phenomenon, it is very important to conserve indigenous plant genetic resources representative of the prevailing genetic diversity especially from under-utilized agro-ecological regions.Field gene banks and in vitro gene banks are playing an important role in this perspective (Ravindran et al., 2005).
Zingiberaceae, known as ginger family, is one of the largest flowering families comprising 53 genera and more than 1200 species (Ahmad, 2010).Members of ginger family notably Curcuma longa and Zingiber officinale have been used for centuries in Chinese, Korean, Japanese and Indian cuisines and traditional Southeast Asian medicines.These species are important natural sources of spices, herbal medicines, dyes, perfumes and multipurpose aesthetic compounds (Chaveerach et al., 2008).
Myanmar, situated in Southeast Asia, is considered as one of the center of origin of Zingiberaceae plants (Ahmad et al., 2009).Myanmar is the habitat of 161 Zingiberaceae species belonging to 21 genera where Curcuma and Zingiber are the main species (Jatoi, 2008).The favorable climatic conditions and habitat diversity are the main reasons of plants genetic diversity in Myanmar (Ahmad et al., 2009).The Zingiberaceae genetic resources of Myanmar have not been well characterized and due to excessive uses and urbanization these valuable native plants need to be collected, characterized and properly conserved.In Myanmar, most of the Zingiberaceae species grow wild or as backyard plantation that demands conservation efforts (Jatoi et al., 2006(Jatoi et al., , 2007;;Ahmad et al., 2009).
Tissue culture is a common tool for conservation and rapid multiplication of asexually propagated species (Anish et al., 2008).Direct in vitro regeneration of explants without passing through callus phase, in order to keep integrity of the genome, offers a mean for conservation of vegetatively propagated plant species through tissue culture.Different in vitro culture protocols have been used for: Z. officinale (Bhagyalakshmi and Singh, 1988;Hosoki and Sagawa, 1977;Rout et al., 2001); Kaempferia galanga (Shirin et al., 2000); C. longa (Salvi et al., 2001(Salvi et al., , 2002)); Curcuma amada (Prakash, et al., 2004;Barthakur and Bordoloi, 1992) and Curcuma xanthorhiza (Mukhri and Yamaguchi, 1986).Tissue culture for in vitro regeneration of the rhizome bud explants have not been previously reported for Zingiber barbatum species growing widely in Myanmar.
Most previously used protocols involved the use of HgCl 2 for disinfection of the explants.HgCl 2 is a serious environmental pollutant and potentially toxic to human and plants (Brandenberger and Maes, 1997;Ma and David, 2006).Ma and David, (2006) reported a disinfection protocol where HgCl 2 was not used for explants disinfection, but this protocol was used for a single species (Z.officinale only).This study reported a disinfection protocol exclu-ding HgCl 2 for explants pretreatment effective for Curcuma, Kaempferia and Zingiber the three very important genera of Zingiberaceae.
In Zingiberaceae, different plant organs have been used as explants; the most successful for direct regeneration are the rhizome buds (Ma and David, 2006;Prakash et al., 2004), but culturing efficiency of buds from "mature rhizomes" (rhizomes obtained after plants are harvested at the end of the season) and "immature rhizomes" (rhizomes obtained while plants are still growing in the field) have not been studied previously.
This study is a continuation of the previous efforts regarding characterization and conservation of Myanmar Zingiberaceae genetic resources (Jatoi et al., 2007;Ahmad et al., 2009).The specific objectives of this research work were; to extend the environmentally safe pretreatment protocol to other species of Zingiberaceae, to establish a single in vitro direct regeneration protocol effective for different species belonging to Zingiberaceae and to report on the culturing efficiency of buds from mature verses immature rhizomes.In addition, this study presented for the first time the in vitro plantlet production system for Z. barbatum.

Plant material
Rhizomes of different Zingiberaceae species were collected from different sources in Myanmar.Detail descriptions of the germplasm are given in Table 1.These rhizomes were sown in pots in Tsukuba Japan; in a non replicated mode the growth period extended to 8 months (from end of April to end of December).The mother rhizomes produced new rhizomes referred here as "immature rhizomes" with new buds on them which were used as explants.At the end of the season plants were harvested in December and the rhizomes obtained referred here as "mature rhizomes", were stored in the green house and were carefully checked for aeration and fungal contamination from time to time.In the month of March when the dormancy period came to an end, the mature rhizomes developed new buds which were also used as explants (Figure 1A).

Rhizomes pre-treatment
The buds from immature and mature rhizomes were used as explants depending upon their availability.The immature rhizomes buds were available in large number but only in the growing season when the plants were young.The mature rhizomes buds were available in small number but for longer time even when the rhizomes were not growing.The rhizomes were soaked in water, scarified and washed carefully.Each rhizome was sliced into small pieces (1 to 3 cm) with buds on them (Figure 1B).The rhizomes pieces were briefly treated with Tween 20 and rinsed with deionized distilled water for 5 min.Later on, the rhizomes were immersed in hot water (50°C) for 10 min.After the hot water treatment, rhizomes were shifted to clean bench and rinsed with autoclaved distilled water 4 to 5 times.Then, the rhizomes pieces were completely submerged in 10% common bleach (pH 12.6) for 30 min and rinsed once with autoclaved distilled water.The buds (5 to 10 mm) were cut apart from the rhizomes pieces.The buds were treated with 0.5% plant preservative mixture (PPM) for 10 min, rinsed in autoclaved distilled water, submerged in 70% ethanol and again rinsed with sterile distilled water.

Culturing conditions
Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), containing 3% (w/v) sucrose and 0.8% (w/v) agar, was prepared with pH 5.6 ± 0.2.The media was microwaved till the agar was dissolved.When the temperature dropped down to 50°C the media was supplemented with a growth regulator, 6-benzyladenine and a commercial fungicide, Benlate (50% of benomyl, methyl 1-(butylcarbamoyl) benzimidazol-2-ylcarbamate) purchased from Sumitomo Chemical Garden Products Inc. Japan.Five ml media was poured into each test tube.The test tubes containing MS medium were autoclaved and later on stored at 4°C for subsequent use.The sterilized buds were cultured in the test tubes containing sterilized MS medium supplemented with benzyl adenine and benlate (Figure 1c).The test tubes were shifted to the growth room with room temperature maintained at 25 ± 1°C with 16 h light and 8 h dark period under 60 to 70% relative humidity in the culture room.

Environmentally safe protocol
The use of metals free pre-treatment protocol is very important to avoid environmental contamination and insure health safety, especially for places where the dumping facilities of metals are not according to the internationally recommended standard.Heavy metals like mercury are known for immunotoxic and neurotoxin properties (Silva et al., 2005).Although, HgCl 2 is a very effective disinfectant in plant tissue culture explant pretreatment protocols, but it was shown in this study that its use is not imminent and can be avoided.
One of the novelties of this work was to establish a single in vitro direct regeneration protocol efficient for different species of Zingiberaceae simultaneously.Figure 2 represents the work flow of the protocol, from rhizomes harvesting to explant culturing, effective for Curcuma, Kaempferia and Zingiber species.Following this protocol bud explants from Curcuma, Kaempferia and Zingiber after sterilization, were successfully established on the MS media and were contamination free even after successive sub-culturing.

Minimizing explants contamination
The immature rhizomes were directly taken from the soil during the growing period of the plants while mature rhizomes were taken from the green house and therefore, needed to be surface sterilized to eliminate the microorganisms present on the surface of the explants.The use of Tween 20 (20%) and sodium hypochlorite solution (5%) effectively controlled the exogenous contamination.Exogenous contamination can effectively be controlled by using surface sterilants and disinfectants (Bajaj, 1989).
Plants harbor endophytic fungi and bacteria which cause serious contamination of in vitro cultures (Sarasan et al., 2006).The explants were passed through a series of pre-treatment steps including hot water and plant preservative mixture (PPM) treatment which helped to minimize the endogenous contamination.Endogenous Harvest the rhizomes from soil at the end of growing period ↓ Remove all the mud and wash with tap water ↓ Treat with sodium hypochlorite solution (5%) ↓ Sun dry and Store in a well aerated place with no direct sunlight ↓ At the end of dormancy period the new buds start emergence ↓ Scarify the rhizomes carefully and wash with tap water ↓ Slice the rhizomes into small pieces (1 to 3 cm) with buds intact ↓ Treat the rhizomes pieces with Tween 20 for 1 min ↓ Rinse with tap water for 5 min ↓ Immerse in hot water (50°C) for 10 min ↓ Shift to clean bench ↓ Rinse with autoclaved distilled water 4 to 5 times ↓ Submerge completely in common bleach (10%) for 30 min ↓ Rinse once with autoclaved distilled water ↓ Cut the buds (5 to 10 mm) apart from the rhizome pieces ↓ Treat the dissected buds with 0.5% PPM for 10 min ↓ Rinse with autoclaved distilled water ↓ Submerge in 70% ethanol ↓ Final rinse with sterile distilled water ↓ Culture the sterilized buds on MS medium in a test tube ↓ Shift the test tubes to growth room contaminants can make their appearance in the culture even after a long period of time (Bajaj, 1989).To address this problem, Benlate (fungicide) and PPM was added to MS media.
The contamination was recorded for all accessions used in this study (Table 2).Bacterial contamination was a lesser problem compared to fungal contamination.The contamination percentage varied with a range of 0 to 39% for different accessions; while, the cultures of some accessions were totally free of contamination (Figure 3).Loc et al. (2005) showed 40% of zedoary explants cultures which were contaminated.In the case of Zingiber, only 10% cultures were contaminated (Ma and David, 2006).In this work, the overall explants contamination  less than 12% for Curcuma, less than 34% for Zingiber and less then 7% for Kaempferia (Table 2).

Bud-explants growth efficiency
The culturing efficiency of buds from mature rhizomes, which were stored for three months in the green house to pass through the dormancy period, versus immature rhizomes, which were obtained directly from the soil during the growth period of the plants, have not previously been studied in Zingiberaceae.When buds from the immature rhizomes were passed through explants pretreatment steps, these buds were unable to response to the MS media and most of them died (data not shown); the regeneration ranged from 5 to 20%.On Accessions No of the germplams used for explant percentge of survival of bud explant

Curcuma Zingiber Kaempferia
Percentage of survival of bud explant  the other hand, when buds from the mature rhizomes were cultured in MS media, after passing through the same pretreatment protocol, they responded amazingly to the media and more than 40 to 95% buds started induction and produced contamination free plantlets (Figure 4).The data in this paper was based on the bud explants from the mature rhizomes.Figure 1 shows the type of buds selected from a mature Curcuma rhizome, the buds induction after culturing on MS media and the development of contamination free plantlet.
A variable number of bud explants both from Curcuma and Zingiber were cultured depending on the availability of the material (Table 3).The Zingiber buds explant started induction after two days of culturing, while it took 4 days for Curcuma explants to start induction on MS media.A total of 152 buds were planted for Curcuma where 115 grew contaminated free and produced plantlets, while the remaining 37 bud explant either did not grow or got contaminated (Table 3).In the case of Zingiber, 132 buds were planted where 76 buds grew contaminated free and made plantlets, while 56 buds did not grow (Table 3).In the case of Kaempferia, only 15 buds were planted where 8 buds grew contaminated free and made plantlets, while 7 buds did not grow (Table 3).In Zingiberaceae, different plant parts including buds, leaf sheets and inflorescence have been used as explants, where some studies reported rhizomes buds producing better results (Ma and David 2006;Prakash et al., 2004).The buds growth percentage varied from 40 to 95% in Curcuma, while this percentage decreased in Zingiber (53 to 75%).The C. amada accession ZO18-1 showed the highest survival percentage (Figure 4).Different stages of in vitro plants production including bud explants induction, shooting, rooting and growth at different days are shown in Figure 5.

Direct in vitro regeneration system for Z. barbatum
It is the first report on the in vitro plantlet production using Z. barbatum buds explants.The same protocol, used for other species in this study, was followed for Z. barbatum in vitro plantlet production (Figure 2).Unlike other species studied, Z. barbatum produced more plantlets (1 to 6) from a single bud where other species produced lesser plantlets (1 to 3) from a single bud in the test tube on MS media.Similarly, Z. barbatum explants survival rate was the highest (75%) among the other accessions of Zingiber (Table 3).The type of bud explants used, stages of shooting and rooting and the fully grown plantlet in the test tube on MS media are depicted for Z. barbatum in Figure 6.

Conservation through direct regeneration
As pointed out by Hussain et al. (2008), it is very important to select the representative diversity germplasm from fields and properly conserve them in vitro.Only a few Zingiberaceae species like turmeric and ginger are cultivated in some areas of Myanmar, while others like Z. barbatum and C. amada grow wild or are kept as backyard plantation.These plants are very essential not only to keep the integrity of the agro-ecosystem, but they are principal sources of traditional medicines.Due to the human activities including both urbanization and excessive use of the native plants without re-plantation, it is not only needed to collect these valuable genetic resources and characterize them but also their proper conservation is essential for their sustainable use.Being vegetatively propagated plants, plant tissue culture is a desirable method for their conservation.Direct regeneration is preferred for conservation through tissue culture in order to minimize soma clonal variation and genotype dependency which are likely to arise in the case of regeneration through callus, Tyagi et al., 2004).Using a single in vitro direct regeneration protocol for different species belonging to different genera of a family, can certainly reduce the cost and time needed for in vitro conservation.This protocol needs a further investigation to check the effect of different concentrations of different hormones in various combinations on the multiplication of the plantlets.

Figure 1 .
Figure 1.Different stages of buds explant culturing, (A) Mature Curcuma rhizome with newly developed buds at the end of the dormancy period; (B), Rhizomes piece possessing the bud explant to pass through disinfection before culturing; (C), a bud explant cultured on MS media; (D), a regenerated plantlet on MS media.The arrows indicate the buds explant on the rhizome.

Figure 2 .
Figure 2. The work chart of the protocol effective for Curcuma, Kaempferia and Zingiber for the in vitro plantlet regeneration from rhizome harvest to in vitro culture.

Figure 3 .
Figure 3.The comparison of the contamination percentages that occurred in the different accessions of Curcuma, Zingiber and Kaempferia after the explants were cultured on the MS media.

Figure 4 .
Figure 4.The comparison of explants survival percentages of different accessions of Curcuma, Zingiber and Kaempferia after the culturing on the MS media.

Figure 5 .
Figure5.The regenerated Curcuma (ZO130) plantlets after buds were cultured on MS media in test tubes; the shooting started in the first week after culturing followed by rooting of the buds (the photographs were taken before changing the media each time).

Figure 6 .
Figure 6.Different stages of Z. barbatum buds explant culturing.(A), a mature rhizome with the arrows pointed to the buds emergence on the rhizome at the end of the dormancy period, These buds were passed through sterilization and then cultured on MS media; (B), emergence, shooting and rooting of the explants; (C), a 50 days old plantlet.

Table 2 .
The contamination details of bud explants of the different accessions infected with bacteria and fungus.

Table 3 .
The number of bud explants cultured from different accessions and their percentage of growth.