Comparison between classical and modern (biotechnology) breedings
Indonesia is the most biodiverse country in the world after Brazil, and its biodiversity is reflected by the vast species of flora found there. The Orchidaceae are one of the largest and most diverse families in the plant kingdom (Arditti, 1992). In Indonesia, orchids are very popular ornamental crops, both as cutting flowers and as potted plants. Many orchid species belonging to the Orchidaceae family can be found only in Indonesia. Most of these species are classified as tropical orchids, which comprise the greatest part of the orchids' diversity. Orchid flowers have fabulous variations based on size, shape, structure, odor, color, and floriferousness.
Indonesian orchids are very unique and exotic. The black orchid (
Micropropagation methods can be applied using biotechnology. Modern biotechnology is important in agriculture, particularly in the economically important horticulture industry, and methods such as genetic transformation have become increasingly important tools for
improving cultivars and studying gene function in plants. This is particularly true in orchids, which are highly valued ornamental plants that are continually being genetically altered. Of the reports published to date on the genetic transformation of orchids, most have focused on transformation techniques targeting specific genes and areas related to crop improvement. We have used the
2. Orchids are an important horticultural plant
Horticulture, literally "garden cultivation", is a branch of agriculture concerned with cultivation of crops that also includes agronomy and forestry. Traditionally, horticulture deals with garden crops such as fruits, nuts, vegetables, culinary herbs and spices, beverage crops, and medicinal, as well as ornamental plants.
In Indonesia, orchids are very popular ornamental crops, and many people enjoy keeping orchids in their homes as decorative plants. On the other hand, orchids such as Dendrobium, Cymbidium, Eulophia, and Habenaria are used in traditional medicine as restoratives and to treat various diseases (Puri, 1970). The market for orchids is worldwide, providing opportunities to grow orchids not only as a hobby, but also for commercial purposes. Horticulturally important species are clustered into taxonomic sections including Phalaenanthe, Spatulata (Ceratobium), Latourea, Formosae (Nigrohirsutae), Dendrobium (Eugenanthe), and Callista (Schelpe and Stewart, 1990). The breeding of orchids is common since most species have high crossability with other orchids, including hybridization between different genera.
3. Orchid breeding
Enhancing productivity and qualitative traits are the main objectives in general horticultural breeding programs. Common goals in breeding new varieties of orchids mainly concern flower size, flower shape, floriferousness, flower color, early flowering, compact growth (dwarfing), resistance to pathogens, and flower longevity. However, breeding programs are successful if the products are marketable regarding extrinsic (color, size, shape) and intrinsic (odor, longevity) traits. Breeding of ornamental plants can be achieved by several methods, either through conventional breeding or biotechnology.
3.1. Classical breeding
Classical or conventional breeding methods using crosses, such as intraspecific and interspecific hybridization of orchid species, are a common way to create new varieties. The French hybrid variety registered in 1934, Dendrobium Pompadour (PPPC), is comprised of three chromosome sets of
Variability in the percentage of viable progeny is generally determined by the genome or cytogenetic relatedness; more similar genome constitutions have more normal meiotic
pairings (Kanemoto and Wilfret, 1980). The development of new F1 hybrids of orchids allows high flower quality to be achieved, which is an important factor not only for hobbyists but also for commercial purposes.
Creating new varieties can be conducted without hybridization. Polyploidization is the simplest way to induce variability in horticultural plants. Chromosome doubling occurs in existing polyploid plants, but it is more likely to produce a positive effect from a diploid base (Levin, 2002). The induction of chromosome doubling is generally performed
3.2. Modern breeding through biotechnology
Plant genetic transformation is potentially a powerful tool for orchid breeding as it can break the species barrier and bring in favorable traits from other gene pools that are not easily accessible through traditional breeding techniques. Transformation of orchids has been accomplished mainly using
|Point of view||Classical breeding||Modern breeding (Biotechnology methods)|
|Source of gene desired||limited||unlimited|
|Insertion of gene desired||indirect||direct|
Wakita and colleagues (2001) reported that the nutritional value of sweet potato was improved by transgenic changes to the fatty acid composition. Transgenic tomato plants expressing the ACC deaminase gene withstand flooding stress (low oxygen) better than untransformed plants and are less subject to the deleterious effects of root hypoxia on plant growth (Grichko and Glick, 2001). The transgenic plants have greater shoot fresh and dry weight, produce lower amounts of ethylene, and have higher amounts of leaf chlorophyll content.
During 1998-2001, more than 40 orchid genes were discovered. However, only a few of these sequenced genes could be directly applied to orchid production and improvement (Kuehnle, 2007). Some of the published orchid genes are summarized in Table 2.
|Gene name||Gene origin||Reference|
|DCAC||Yang et al., 1996|
|DOH1||Yu and Goh, 2000|
|DOMAD1||Yu and Goh, 2000|
|Yu and Goh, 2000|
Yang et al., 2003
Using the orchid genes or other sequenced genes, genetic transformation of orchids can be conducted using three major approaches including particle-bombardment, direct gene transfer to protoplasts, and
|Semiarti et al., 2007|
Semiarti et al., 2010
|Belarmino and Mii, 2000|
Chin and Mii, 2011
|Semiarti et al., 2008|
|Particle-bombardment||Kuehnle and Sugii, 1992|
|Semiarti et al., 2009|
|Particle-bombardment||Yang et al., 2000|
3.3. Cultivation and propagation
Another modern biotechnological procedure in orchid breeding is plant tissue culture, which is widely used for orchid cultivation and propagation. The orchid seed has no endosperm, so the probability of growing naturally in its habitat is very low, necessitating plant tissue culture. Plant tissue culture techniques use rich nutrient culture medium and aseptic conditions with growth in transparent bottles. The orchid seed obtains its nutrients from the media and can grow normally. The phenotype of the plantlet varies from its parent. If we use explants from leaves, shoots, or other vegetative organs, we can produce identical offspring. As we know, orchids need a long time to reproduce, but using plant tissue culture techniques, we can produce offspring in large quantities in a very short time. This is very useful for breeders and orchid conservationists. Plant tissue culture is also a tool that facilitates genetic engineering, so it is being continuously refined and should be studied by orchid researchers.
3.4. Standard techniques of orchid micropropagation
Several aspects that need to be considered in plant tissue culture are culture conditions, media components, and aseptic conditions. Ideal culture conditions for orchid micropropagation depend on the type of medium, pH, illumination, photoperiod, and temperature. Both liquid and solid media can be used, but proliferation is faster and more extensive in liquid media; however, differentiation is always better in solid media. The optimum pH for orchid micropropagation is approximately 5-7. Suitable illumination for orchid tissue is from darkness to 2000 ft-c (ft-candles). Photoperiods used for orchid tissue culture vary from none (constant illumination or darkness) to short and long days (12-18 hours). Orchid tissue culture is usually maintained at a temperature of 22 to 26°C (Arditti and Ernst, 1993). The medium components for orchid micropropagation are mostly the same as those for other plant culture media. It must contain macro elements (C, H, O, N, S, P, K, Ca, Mg, and Fe), microelements, hormones, myo-inositol, vitamins, amino acids, adsorbents, solidifying agents, and complex organic additives (Arditti and Ernst, 1993; George and Sherrington, 1984).
In principle, all plant cells can be cultured because they all have totipotency and autonomic properties. However, in practice, only meristematic cells can be used as explants for plant tissue culture. Micropropagation of orchids was first performed by German orchid expert Hans Thomale in 1957, who used
Orchids, like any other plant cultured in medium, need acclimatization before being planted in a pot. Plant tissue culture uses rich media to support orchid growth, which while good for the orchid, has the effect of limiting its ability to produce food for itself. Acclimatization is needed to adapt the orchid to its new environment and to change it from heterotrophic into autotrophic conditions (Arditti and Ernst, 1993). In their natural habitat, orchids cannot always obtain optimum conditions to grow, so we need to adapt orchids to their new environment slowly. This is usually done in a greenhouse.
Agrobacterium-mediated genetic transformation of Indonesian orchids
4.1. Transformation of
Phalaenopsis amabilis (L.) Blume
To improve the potential of the orchid’s micropropagation, we developed a genetic transformant of
To introduce the
Transformation was started by preparation of overnight cultures of
Genomic DNA from the putative 35S::
|Exp||Number of protocorms examined||Number of protocorms producing shoots||Frequency of transforma-tion (%)*||Number of|
In addition, both
Although the function of members of the class 1
Maryani (2010, personal communication) introduced the
The core component of genetic modification of orchids is the need to create efficient and reproducible gene transformation systems. A reproducible methodology for the genetic transformation of orchids, and better recognition of the factors affecting the transformation process, are needed in order to support this objective. Previous studies have reported orchid transformation either directly through the delivery of marker genes such as those encoding
In order to improve the frequency of
To determine the growth rates of orchid embryos and protocorms, the sizes, colors, and shapes of the embryos or protocorms were evaluated as described by Dressler (1981). At Stage 0, each intact seed (270–400 µm long) with its embryo (100–200 µm long) is coated by a layer of net-like cells, the testa. At Stage 1, the testa spreads apart and the embryo swells into an ovoid-shaped mass of cells. At Stage 2, the seed coat cracks and the mass of cells grows outside the coat (0.5–1.0 mm long). At Stage 3, the mass of cells elongates gradually into a cone-shaped body (1.0–1.4 mm long). At Stage 4 (the protocorm), root hairs emerge from the basal portion of the cone-shaped body, which turns green. At Stage 5, the photosynthetic protocorm forms a leafy shoot at its apex and forms new root hairs. After Stage 5, seed germination is complete, two leaves gradually emerge, and roots are formed.
4.2. Plasmid vector and bacterial strain
Using the binary plasmid vector pBI121 (Clontech Laboratories Inc., Otsu, Japan), containing a kanamycin resistance gene and the 35S CaMV promoter with the 3'
4.3. Transformation and transformant regeneration
Overnight cultures of
4.4. DNA analysis by Southern hybridization
Genomic DNA from 9-month-old leaves of five independent transgenic lines of
4.5. Effect of tomato extract on the formation of shoots from protocorms of
We tested coconut water and tomato extract as potential supplements to accelerate the growth of
We also analyzed growth rates on NP medium with or without coconut water and tomato extract (Figure 6A). The fastest rate of embryo development was observed on NP medium supplemented with both coconut water and tomato extract. Protocorms cultured on NP medium containing tomato extract alone appeared to change from yellow to green more rapidly than those cultured on NP medium containing coconut water alone. Tomato extract thus appeared to affect the growth rate at all stages of embryo development, including the
|Concentration of tomato extract|
|Total no. embryos examined||Experiment No.||Embryo|
Stage 0 (%)
Stage 3 (%)
|Green protocorm Stage 4 (%)||Protocorm with shoot apical meristem Stage 5 (%)|
|Component||Coconut water||Tomato extract|
formation of the shoot apical meristem prior to the emergence of the leaf primordia. The tomato extract contained carotene, vitamin C, and other anti-oxidants which were not detected in coconut water (Table 6). These components could affect the growth of the embryo.
Oladiran and Iwu (1992) showed that fully-ripe tomato fruit contains basic nutrients and essential vitamins, as well as trace elements. Among these, carotenoids with cyclic end-groups were essential components of all photosynthetic membranes and played several roles, including protection against photo-oxidation (Cunningham et al., 1996). These are potential candidates for the growth-promoting compounds in the tomato extract, as it is rich in carotenoids. We therefore tested a single carotenoid, lycopene, for its possible effects on growth promotion, but found no significant effect at concentrations typically found in tomato extracts [≤ 0.1% (w/w)], while high concentrations of lycopene inhibited seed growth (data not shown). Further studies on other components found in tomato extract are needed to determine whether any single compound has an effect, or if several compounds have a synergistic effect, on the growth and development of
4.6. Effect of pre-culture of protocorms on NP medium containing tomato extract on the transformation frequency of
P. amabilis orchids
Protocorms were pre-cultured on NP medium supplemented with coconut water and/or tomato extract prior to transformation to determine the effects of pre-culture supplementation on the frequency of transformation (Table 7). The transformation efficiency was determined based on the percentage of protocorms that produced shoots on the selective medium out of the total number of protocorms examined. The transformation frequency of regenerated shoots was increased from 1.2% on NP medium with coconut water alone to 13.2% on NP medium containing 100 mg l-1 tomato extract alone, and to between 6.8–16.6% on NP medium containing both coconut water and tomato extract (Table 7; Figure 6B, panel D). These results were higher than the frequency of transformed regenerated shoots on medium containing coconut water alone (1.2%; Table 7), confirming the observations made by Semiarti et al. (2007).
In the case of transformation with pBI121-p35S::GFP, transformed regenerated shoots were produced at frequencies of 9.8–13.5% following pre-culture on NP medium supplemented with both coconut water and tomato extract (Table 7). Overall, the transformation frequencies of protocorms pre-cultured on NP medium supplemented with tomato extract alone, or with both coconut water and tomato extract, were higher than those for protocorms pre-cultured on NP medium supplemented with coconut water alone, suggesting that the growth rate of protocorms was related to the pre-culture conditions which are therefore important for the regeneration of transformed shoots.
Several studies have examined the use of rich sources of nutrients, vitamins, and phytohormones, including coconut water, carrot, maize, or potato extracts, as possible supplements for stimulating the germination of various orchid species (Arditti and Ernst, 1993; Raghavan, 1997; Islam et al., 2003; Mishiba et al., 2005; Chansean and Ichihashi, 2007). More studies on other sources of nutrients may be required to establish an optimum method for transformation.
|Total no. protocorms examined||No. protocorms producing shoots (% of total)|
|pBI121 (vector)||+||-||1200||14 (1.2%)|
|pBI121 (vector)||-||+||1200||159 (13.2%)|
We used this transformation method on the pandanus orchid (
4.7. Molecular analysis of putative transformants
We examined the genomic DNA from
transformation with the plasmid pBI121-p35S::GFP were examined for the presence of the
To confirm the presence of the
For further analysis, we purified total poly(A)+ RNA from individual leaves of an untransformed wild-type plant, a plantlet transformed with pBI121, and three lines transformed with pBI121-p35S::GFP. We quantified the relative levels of
4.8. Transformation of
Vanda tricolor Lindl. var. Suavis
Three days before infection, eight week-old germinated protocorms were transferred onto fresh NP medium containing 1 µl l-1 of 2,4D.
Three days after maintaining the protocorm in the bacterial-elimination medium, protocorms were washed with ½ NP liquid medium containing 20 mg l-1 meropenem three times, then were transferred to selection medium (SIM with 300 mg l-1 kanamycin and 8 mg l-1 meropenem). Protocorms were mantained in this medium for 5 weeks and subcultured every week or less to eliminate
The kanamycin test was performed twice on the protocorms of
From the study of Dwiyani et al. described above, two points can be infered: first, the addition of AS into co-cultivation medium is required. The addition of AS into the inoculum increased the percentage of protocorms surviving in the selection medium above that with the addition of AS into co-cultivation only. AS supplementation in this step of transformation (inoculation) might stimulate higher concentrations of AS within the tissues of the treated protocorm, thereby eliciting higher
The resistancee of
4.9. Transformation of the black orchid (
Coelogyne pandurata Lindley)
The black orchid (
4.10. Developmental phases of the black orchid (
Coelogyne pandurata Lindley) embryo
The development of black orchid embryos during seed germination can be classified into six phases based on growth and morphology: phase 1) yellowish embryo, phase 2) green embryo, phase 3) bipolar embryo, phase 4) first leaf formed embryo, phase 5) second leaf formed embryo, and phase 6) third leaf formed embryo. The time-course of embryo development shows that the embryo starts to change from yellowish (phase 1) into green (phase 2) at one to two weeks after sowing. At three to four weeks, the green embryo forms a bipolar structure (phase 3), with one side darker than the other. The darker pole of the embryo changes into leaf primordia (phase 4) at the fifth week, a protocorm with two leaves at seven weeks (phase 5), and a protocorm with three leaves at eleven to twelve weeks (phase 6) (Table 8).
At twelve weeks after sowing, based on the growth rate of embryos, the data revealed that ½ NP medium is the best to support and accelerate growth that will result in seed germination. Approximately 86% of protocorms grew up to phase 5 (Table 8). The results show that for embryo development during seed germination in black orchids, a half-strength concentration of complete element-containing medium is the best. It might be that the content of macro- and micro-elements in the half strength medium provides a suitable consentration to promote the development of the embryos, so it is not necessary to use full-strength basic medium. As described by Arditti and Ernst (1993), tissue culture is an empirical science. It is difficult to predict the type of explant, media, and conditions that are suitable for a specific genus or species or clone. It is not possible to explain why certain media and culture conditions lead to success while others fail. In the black orchid, the half-strength NP medium may be the best for seed germination, so that the embryo will respond better to genetic transformation than that if we used full-strength NP medium.
|Variation of Medium||No of seeds||Percentage of growing embryos at each phase||Death of protocorms|
|Phase 1||Phase 2||Phase 3||Phase 4||Phase 5||Phase 6|
4.11. Insertion of the
KNAT1 gene in the black orchid
Genetic transformation of plasmid 35S::KNAT1 and pGreen vector into orchids was carried out according to the method of Semiarti et al. (2007), except that the liquid medium used to rinse the protocorm was half-strength NP medium with 300 mg l-1 cefotaxim. SIM (Shoot Induction Medium; 0.15 μM NAA + 5 μM 2-IP) supplemented with 100 mg l-1 kanamycin for selecting independent transformants. Into each step 75 mg l-1 acetosyringone was added to improve the efficiency of T-DNA insertion as described by Semiarti et al. (2010). The frequency of transformation was measured as the ratio of the number of surviving protocorms per total number of transformed protocorms (Table 9).
|No.||Treatment||Kan, 300 mg l-1 (+/-)||No. of protocorms||Percentage of Kan-resistant plants|
The expression of the
transformants. Further results of transformation with
5. Conclusions and future prospects
We thank all students who work in the laboratory of Plant Tissue Culture, Faculty of Biology Universitas Gadjah Mada. This research was supported in part by the Indonesian DGHE Research Competition grant HB XVII 2009-2010 and the Japanese Academic Frontier Research Grant to CM from 2005-2010. We thank the Bunga Rintee Orchid Nursery for the gift of black orchid fruit and to Mr. Wirakusumah for the gift of 4-month-old protocorms and for valuable discussion on black orchid culture techniques.
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