Gene transfer in Allium: recent developments and future prospects




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Gene transfer in Allium: recent developments and future prospects




Si-Jun Zheng, Betty Henken, Frans Krens & Chris Kik

Plant Research International, Wageningen University and Research Center,

P. O. Box 16, 6700 AA Wageningen, The Netherlands

Abstract

This review outlines recent developments of gene transfer systems in Allium crops. Gene transfer can be realized via sexual hybridization, somatic hy(cy)bridization and genetic transformation. Sexual hybridization has always been an important tool for the introduction of genetic variation needed for plant improvement. In this context the introduction of wild germplasm into cultivated crops is highly important as much agronomically important traits, like disease and pest resistance, the genetic variation needed is often not found in the cultivated species. In Allium alien introgression research has predominantly been carried out in onion and to a lesser content in leek. No alien introgression research has been carried out for garlic. The latest developments in this area, like the gene pool approach to introgress agronomically important traits from wild relatives in onion, and the possibilities for sexual hybridization in garlic will be discussed.

If sexual hybridization is not possible, somatic hy(cy)bridization and genetic transformation can provide a way forward. Somatic hy(cy)bridization was successfully used to transfer cytoplasmic male sterility (CMS) from onion to leek and also in bridging the gap between onion and garlic. However until present the perspectives of this technique in Allium breeding are not clear. Recently a breakthrough was made in opening up genetic transformation technology for Allium crops. Both Agrobacterium-mediated and biolistic gene transfer systems have been developed in onion, shallot and garlic. Currently transgenic Allium plants are available harbouring bar or acetolactate synthase (ALS) genes for herbicide resistance, Bt genes for insect resistance and antisense alliinase genes for a milder taste. Transgenic onion and shallot plants proved to have a normal phenotype, were fertile, had a normal ploidy level and inherited their genes in a Mendelian fashion. As a last topic in this review safety assessment of genetically modified (GM) Allium crops is discussed.


Introduction

Onion (Allium cepa L. group Common Onion), shallot (A. cepa L. group Aggregatum) and garlic (A. sativum L.) are very important vegetable crops worldwide. They are members of the genus Allium (Family Alliaceae), a genus which comprises about 750 species (Fritsch & Friesen, 2002). Onion is cultivated mainly as an annual but some types are treated as perennials. It is propagated by seeds, bulbs, or sets (small bulbs). Onion bulbs are quite variable with respect to shape, size, skin and flesh colour, pungency, skin retention, storage ability and dry matter content. Shallot differs from common onion primarily in bulb characteristics. The bulbs of common onion are large, normally single, and plants are grown from seeds or from seed-grown sets. The bulbs of shallot are smaller compared to the bulbs of common onion, they form an aggregated cluster of small bulbs as a result of the rapid formation of lateral bulbs or shoots. Cultivation of shallot is predominantly vegetatively via daughter bulbs (sets). Two types of onion cultivations exist namely cultivars based on F1 hybrid breeding (F1 hybrids) and cultivars based on mass and family selection (OPs; Kik et al., 1998). Nowadays F1 hybrids are increasingly dominated the world seed market of onions. For shallot most cultivars are OPs although in recent years also F1 hybrids are on the market. Garlic is favoured throughout the world for its very specific flavour and is used in most kitchens in the world as an important spice. Furthermore, garlic is known for its therapeutic and medicinal use. Garlic is a perennial monocot that is propagated vegetatively via cloves and bulblets. Garlic breeding via sexual hybridization is still in its infancy. Thus, garlic breeding relies on simple selection and multiplication of existing germplasm.

Onion, shallot and garlic are vulnerable to a number of diseases, pests and viruses (Rabinowitch, 1997). In temperate zones Botrytis and Fusarium diseases, downy mildew (Peronospora destructor), white rot (Sclerotium cepivorum), thrips (Thrips tabaci) and onion fly (Delia antiqua) can cause substantial yield losses. In tropical zones purple blotch (Alternaria porri), anthracnose (Colletotrichum gloeosporioides) and beet armyworm (Spodoptera exigua) are threatening the onion and shallot cultivations. Because there was no reliable resistant sources available in onion and shallot germplasm analysed (Zheng et al., 2000), it was not possible to carry out conventional breeding programme to produce resistant cultivars. Almost all commercial garlic cultivars have been shown to be infected with a complex of viruses that include leek yellow stipe virus (LYSV), onion yellow dwarf virus (OYDV), shallot (garlic) latent virus (SLV, GLV), garlic common latent virus (GCLV) and garlic viruses (GarVs)A-D (Salomon, 2002). In view of the aforementioned problems gene transfer methodology is of pivotal importance for the development of better-adapted cultivars of onion, shallot and garlic.

Improvements in gene transfer technology for Allium species have been remarkable in the past few years, especially in the area of genetic transformation. Therefore, in this review particular attention will be paid to this type of gene transfer in Allium crops and safety issues related to GM Allium crops will be briefly discussed.


Gene transfer via sexual and somatic hy(cy)bridization
Species hybridization for plant improvement has always been an important tool for the introduction of genetic variation in the breeding of new cultivars, as wild relatives of cultivated species contain gene reservoirs for agronomically useful traits. With respect to sexual hybridization it is known already for a long time that the transfer from genes from one species to another species can be difficult. This proved to be also true for Allium (for review: Kik, 2002). However an important breakthrough in this area has recently been accomplished, namely Khrustaleva & Kik (1998, 2000). They showed that the hybridization barrier between A. fistulosum and A. cepa can be overcome using A. roylei as a bridging species. This results in highly segregating so-called bridge-cross populations [A. cepa X (A. fistulosum X A. roylei)]. Especially the presence of many disease and pest resistances in A. fistulosum, but also in A. roylei, makes this gene pool approach very attractive for plant breeding purposes. From a fundamental point of view these bridge-crosses are also very interesting: they showed for example that differences in repetitive DNA between onion, A. fistulosum and A. roylei are very large, furthermore they showed that randomly distributed chiasma, as can be found in A. cepa and A. roylei, are dominant over proximally localized chiasma, which are present in A. fistulosum. The next step in this alien-introgression research will be to our understanding of the transmission of chromatin from wild species into the cultivated species. What factors influence this process? Can genes involved in species incongruency or in nuclei-cytoplasmic interactions be located and identified? All the techniques needed to study these questions, e.g. molecular marker (van Heusden et al., 2000a, b) and in situ hybridization technology (Khrustaleva & Kik ,1998, 2000), have been developed for Allium.
The possibilities of using sexual hybridization in garlic improvement are becoming increasingly realistic nowadays. Etoh et al. (1988) reported that they obtained 19369 garlic seeds via sexual hybridization. Jenderek et al. (2000) evaluated 64 accessions and found 36 accessions producing seeds. Evaluation of other characteristics influencing fertility as well as the search for new germplasm is underway by many groups, for example Kamenetsky and Rabinowitch (2001) found at least four morphological types differing in flower/topset traits in garlic germplasm.
Somatic hybridization can also be used to transfer gene from one species to another, especially in those cases where sexual hybridization is no option. For example, somatic hy(cy)bridization has been used to transfer cytoplasmic male sterility (CMS) from onion to leek to establish the basis for F1 hybrid breeding in leek. It appeared possible to produce both symmetric and asymmetric protoplast fusion products between onion and leek (Buiteveld et al., 1998a,b). Although the asymmetric fusion products, with inactivated onion nuclear DNA, were not able to regenerate plants, the symmetrically fused calli yielded high numbers of regenerated plants. Most of the plants were actual hybrids, as was shown by flow cytometric analysis of nuclear DNA and genomic in situ hybridization. However, a number of these hybrids had an aneuploid background. All somatic hybrids were phenotypically intermediate in between both parents. Shimonaka et al. (2002) reported the production of somatic hybrid plants between Japanese bunching onion (A fistulosum L.) and bulb onion (A. cepa L.) via protoplast electrofusion. Plant regeneration was achieved in 33 out of 325 (10.1%) calli. Some regenerated plants expressed abnormalities, but two were successfully transplanted in a greenhouse. Cytogenetical and DNA analyses revealed these two regenerants to be amphidiploids (2n=4x=32). Furthermore, it was shown that another three regenerants possessed the nuclear genome of Japanese bunching onion, whereas, their chloroplasts were from bulb onion.
Gene transfer via genetic transformation

Regeneration system

The development of an efficient system for genetic transformation is a valuable extension of the gene transfer tools for further crop improvement. In order to establish a successful Allium genetic transformation system, two key factors should be taken into account. One is the development of sophisticated methods to recover intact plants, either from fully dedifferentiated tissue or from organized tissues that are easy to regenerate. The other is the refinement of methods for the introduction of exogenous DNA into Allium germplasm.

In Allium, various plant regeneration systems have been developed using different starting material. Eady (1995) reviewed the different source materials used for in vitro culture of Allium species. The most successful regeneration systems in Allium use (im)mature embryos, root tips (segments), flower buds, suspension cultures or protoplasts as starting materials. A key point in Allium regeneration protocols is the fact that tissues are used consisting of actively dividing cells, such as mature or immature embryos of onion and shallot or calli induced from apical and non-apical root segments of in vitro plantlets of garlic (Eady et al. 1998; Zheng et al. 1998, 1999, 2003). Such young and actively dividing callus material is uniquely suitable for genetic transformation.
Transformation system
Transformation of recalcitrant monocots like rice, wheat, barley and maize has been achieved by using direct gene transfer systems: chemical methods, electroporation, particle bombardment and silicon carbide fibres. Recently, Agrobacterium-mediated transformation of monocots has gained favour and many transgenic plants have been obtained using specific Agrobacterium strains ( Hiei et al., 1994; Rashid et al., 1996; Cheng et al., 1997; Tingay et al., 1997; Ishida et al., 1996; Arencibia et al., 1998). Agrobacterium-mediated transformation offers several advantages over other systems, the most important being the capability of delivering a single or low number of intact copies of relatively large segments of foreign DNA. Nowadays Agrobacterium tumefaciens-mediated transformation is routinely utilized in gene transfer to monocotyledonous plants, such as rice and maize (Hiei et al., 1997).

Recently, reports were published showing that genetic transformation has become possible in Allium and this without any doubt is a major step forward (for review, Eady 2002). The progress of transformation research in Allium in the last 10-15 years is summarized in Table 1. With report to particle bombardment, Klein et al. (1987) developed as the first one a high-velocity microprojectile method and demonstrated that epidermal tissue of onion could take up foreign DNA sequences. Wang (1996) obtained transgenic leek plants by particle bombardment with the barnase and barstar genes, and it was shown that the genes were present in the leek genome. Transient expression was shown with particle bombardment in garlic (Barandiaran et al. 1998; Ferrer et al. 2000). Park et al (2002) and Sawahel (2002) reported that transgenic garlic plants were generated by particle bombardment. With respect to Agrobacterium mediated gene transfer, Dommisse et al. (1990) demonstrated that onion is also a host for Agrobacterium as evidenced by tumorigenic responses and opine production inside these tumours. Eady et al (2000) developed a stable transformation protocol using immature embryos of A. cepa via Agrobacterium tumefaciens. Kondo et al. (2000) used highly regenerative calli derived from shoot primordial-like tissues to produce transgenic garlic plants by Agrobacterium-mediated gene transfer. Zheng et al. (2001a, 2001b, 2004a, 2004b) developed a reproducible and stable transformation protocol using calli derived from mature embryos of onion and shallot or using calli derived from apical and non-apical root segments of in vitro plantlets of garlic via Agrobacterium tumefaciens. Due to the aforementioned efforts in developing reliable transformation systems for Allium crops nowadays transgenic shallot and garlic plants containing Bt resistance genes have been produced which confer resistance to beet armyworm (Spodoptera exigua) (Zheng et al. 2004a, 2004b). Furthermore, transgenic plants containing herbicide resistance and antisense versions of alliinase genes are available (Eady 2002).


Safety assessment of genetically modified (GM) Allium crops
Genetic engineering of plants to produce genetically modified organisms (GMOs) was initiated nearly three decades ago with the intent to develop improved foods and medicines. In the year 2001, there were about 50 million hectares of GM crops grown worldwide (James 2001). Using GM technology transfer of genes into crops from any class of living organism has become a reality thereby producing novel kinds of crops. Because of this, there is international agreement that a comprehensive safety assessment should be carried out before GM crop plants are grown commercially in agriculture (Dale & Kinderlerer, 1995). As transgenic herbicide and insecticide resistant Alliums have become a reality, it becomes important to analyze their safety aspects. We will try to do this using transgenic Bt shallots and garlic as an example. To answer the political impact of GM crops a number of questions can be raised:

  1. How does the introduced gene changes the modified crop? Based on our experience, the phenotypic appearance of the crop due to the Bt transgene does not significantly change.

  2. Is there evidence of toxicity and/or allergenicity? This question has not been studied yet, but based on the experience with Bt corn and soybean it is not been expected.

  3. What are the effects on friendly organisms in the environment? This is not yet clear as field experiments have to be carried out. But large effects are not be expected based upon the experience gained from corn and soybean.

  4. Does Bt Alliums induce weediness in agricultural habitats or invasiveness in natural habitats? This does not seem to be a real problem as Bt shallots and garlic because these transgenics are only modified into the direction of resistance to beet armyworm. As it is not known from literature that beet armyworm is involved in weediness or invasiveness, we do not expect major problems in this respect.

  5. Does gene flow between Bt Alliums and wild relatives exist and does it pose a threat? Gene flow between Allium species does exist, but it is very limited under greenhouse controlled condition (for review, Kik, 2002). Flowering of Bt shallots and garlic under current cultivation practice occurs sporadically. Therefore, the risk of gene exchange is very limited.

Insecticides based on endotoxin proteins of Bacillus thuringiensis have been in use for 40 years, and have a safety record for non-target invertebrates and vertebrates including mammals that far surpasses that of any synthetic chemical insecticide. This safety record, combined with the efficacy of certain Bt Cry proteins and the advent of recombinant DNA technology, led to the development of transgenic insect-protected Bt crops that are being adopted rapidly, especially Bt cotton and Bt maize, by farmers in the United States and a few other countries (Federici, 2002). From the current state of knowledge, the impact of free DNA of transgenic origin is likely to be negligible (Dale et al. 2002). There is no reason at present to think that these crops present risks greater than those associated with the consumption of non-Bt crops. In fact, Bt crops may be safer for human consumption than conventional crops because they cobtain lower levels of mycotoxins and residues of chemical insecticides. All in all, GM safety issue in Bt garlic and shallots seem to be minimal. However only field and greenhouse experiments will give the crucial data needed to answer these aspects and make these transgenics acceptable for the general public.




Conclusions and future prospects

Gene transfer systems have been developed in Allium crops. Both Agrobacterium-mediated gene transfer and biolistic gene transfer systems can be used to produce genetically modified Allium crops. Already herbicide and insecticide resistant Allium crops have been developed. These transgenics could represent an important stepward to reduce the level of herbicide and pesticide used in Allium cultivations. For a successful introduction of these Allium transgenics in agriculture, however, a through safety assessment has yet to be carried out. A promising next step in GM Allium research is the development of Allium crops with a modified sulphur, carbohydrate or flavonoid metabolism in order to boost the development of tailor-made cultivation for the increasing expanding functional food market world-wide.




Acknowledgements

This research is carried out in the frame work of the European Union project 'Garlic and Health' which is partially financed by an EU FP 5 grant in the area of key action 1 (QLK1-CT-1999-498; www.plant.wag-ur.nl/projects/garlicandhealth).



References
Table 1. Genetic transformation research in different Allium species


Species

Target tissue

Transformation method

Result

Reference

A. cepa

Epidermal tissue

High-velocity microprojectiles

Transient expression of a foreign gene (cat gene)

Klein et al., 1987

A. cepa

Epidermal tissue

High-velocity microprojectiles

Transient expression of GFP

Scott et al., 1999

A. cepa

Bulb

Agrobacterium tumefaciens,

A. rhizogenes, A. rubi

Tumorigenic response and opine production

Dommisse et al., 1990

A. cepa

Zygotic mature embryo after in vitro culturing for 12 days

Agrobacterium tumefaciens

Transient expression of gusA

Joubert et al., 1995

A. cepa

Microbulbs from germinating mature seeds, immature embryo after in vitro culturing for 14 days

Particle bombardment, Agrobacterium tumefaciens

Transient expression of gusA

Eady et al., 1996

A. cepa

Immature embryo

Agrobacterium tumefaciens

Stable expression of nptII and m-gfp5-ER

Eady et al., 2000

A. cepa

Calli derived from mature embryo

Agrobacterium tumefaciens

Stable expression of gusA and hpt

Zheng et al., 2001a, 2001b

A. cepa

Immature embryo

Agrobacterium tumefaciens

Stable expression of bar and antisense versions of alliinase genes

Eady et al., 2002, 2003a, 2003b

A. cepa

Calli derived from mature embryo

Agrobacterium tumefaciens

Stable expression of gusA, hpt and Bt genes (cry1Ca or H04)

Zheng et al., 2004b

A. porrum

Embryogenic callus derived from shoot base

Biolistic system

Stable expression of gusA and bar

Wang, 1996

A. sativum

Leaf, immature bulb, callus from basal plate

Biolistic system

Transient expression of gusA

Barandiaran et al., 1998

A. sativum

Embryogenic calli, leaves and basal plate discs

Biolistic system

Transient expression of gusA

Ferrer et al., 2000

A. sativum

Highly regenerative calli derived from shoot primordial-like tissues

Agrobacterium tumefaciens

Stable expression of gusA and hpt

Kondo et al., 2000

A. sativum

Calli derived from apical meristem of cloves

Biolistic system

Stable expression of gusA, hpt and acetolactate synthase gene (ALS)

Park et al., 2002

A. sativum

Calli derived from apical meristem of immature cloves

Biolistic system

Stable expression of gusA and hpt

Sawahel, 2002

A. sativum

Calli derived from apical and non-apical root segments of in vitro plantlets

Agrobacterium tumefaciens

Stable expression of gusA, hpt, gfp or Bt genes (cry1Ca or H04 )

Zheng et al., 2004a







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