Millets genetic engineering: the progress made and prospects for the future

Millets comprise a highly variable small-seeded group of Poaceae members that can grow in extreme environmental conditions of drought, high temperature and low soil fertility hence, recognized as climate-resilient. Among millets, the phylogenetic closeness of Setaria with other agronomically important grasses like maize, sugarcane, and sorghum helped in its adoption as a translational model plant. Established efficient gene transfer methodology is a prerequisite for embracing plant species as models. However, genetic engineering of some of the economically important millets has been started in the 1990s, but inadequate progress made this group lag behind other members of Poaceae as rice, maize and wheat. Genetic transformation in millets has generally been achieved by a physical method of microprojectile bombardment, recently Agrobacterium-mediated gene transfer technique has also established in some of the millets but with very few reports. The central hindrance in millet transformation is its recalcitrant nature to regeneration through tissue culture techniques. Optimization of highly efficient regeneration procedure for each millet species is thus, necessary to establish advanced transformation system for them. The possibility of alternative transformation approaches is also discussed. The establishment of robust gene transfer methods whether it’s conventional in-vitro tissue culture dependent or in-planta are important for functional validation studies and would enable development of crop improvement strategies. This review presents the progress made on millet genetic transformation, discussing the major challenges that need to be overcome and future opportunities of transgenic techniques in various millets.

     

Isozymic classification of pearl millet (Pennisetum glaucum,Poaceae) landraces from Niger (West Africa)

The principal landraces of the pearl millet,Pennisetum glaucum (L.)R. Br., from Niger have been analysed for their genetic structure at eight enzyme systems coded by 12 loci and 46 alleles. Three groups have been identified: (1) early-maturing pearl millets, cultivated between 8° and 13°E longitude, including the oases from Aïr mountains; (2) early-maturing millets situated more to the west (1° and 8°E longitude), and (3) late-maturing millets. Group 1 shows the highest isozyme diversity. The differences between the accessions represent 8.8% of the total diversity and the differences between the three groups 4.5%. The accessions from groups 1 and 3 are the least distant. When considering pearl millets from areas outside Niger, the chadian and sudanese millets are enzymatically close to the Niger group 1. The pearl millets from Niger group 2 are close to millets from east Mali, northern Burkina Faso and Senegal, and the Niger group 3 to the late-maturing millets group from West Africa. This study should help breeders to select the landraces for improvement and parents for crosses from cultivars of Niger and introduced germ plasm.     

Genetic and Genomic Resources of Small Millets

Small millets are very promising agricultural entity to ensure global food security. They gained remarkable importance in agriculture due to their resilience to climatic changes and increasing demand for nutritious food and feed. The genetic variability in the core and mini-core germplasm of small millets was characterized for nutritional composition and capacity to tolerate abiotic stresses that can be infused in breeding programs. Other than the foxtail millet, availability of genomic information in small millets is far below the mark for use in marker-assisted breeding and other genetic improvement programs. The genome sequence of foxtail millet has recently triggered a plethora of post-genomic analysis and envisaged foxtail millet as a model organism for the C₄ grasses and bioenergy research. Recent developments in the next-generation sequencing technologies enabled us, with the simultaneous discovery of high-throughput markers and multiplexed genotyping of germplasm, to speedup marker-assisted breeding. In this context, an in-depth analysis of the wealth of diverse germplasm resources and future perspectives of integrating genomics in genome-wide marker-trait association and breeding in small millets is worthy.     

Genetic transformation of wheat: progress during the 1990s into the Millennium

Wheat transformation technology has progressed rapidly during the past decade. Initially, procedures developed for protoplast isolation and culture, electroporation- and polyethylene glycol (PEG)-induced DNA transfer enabled foreign genes to be introduced into wheat cells. The development of biolistic (microprojectile) bombardment procedures led to a more efficient approach for direct gene transfer. More recently, Agrobacterium-mediated gene delivery procedures, initially developed for the transformation of rice, have also been used to generate transgenic wheat plants. This review summarises the considerable progress in wheat transformation achieved during the last decade. An increase in food production is essential in order to sustain the increasing world population. This could be achieved by the development of higher yielding varieties with improved nutritional quality and tolerance to biotic and abiotic stresses. Although conventional breeding will continue to play a major role in increasing crop yield, laboratory-based techniques, such as genetic transformation to introduce novel genes into crop plants, will be essential in complementing existing breeding technologies. A decade ago, cereals were considered recalcitrant to transformation. Since then, a significant research effort has been focused on cereals because of their agronomic status, leading to improved genetic transformation procedures (Bommineni and Jauhar 1997). Initially, the genetic transformation of cereals relied on the introduction of DNA into protoplasts and the subsequent production of callus from which fertile plants were regenerated. More recently, major advances have been accomplished in the regeneration of fertile plants from a range of source tissues, providing an essential foundation for the generation of transgenic plants. This review summarises procedures, vectors and target tissues used for transformation, high-lights the limitations of current approaches and discusses future trends. The citation of references is limited, where possible, to the most relevant or recent reports.     

Evidence from RAPD markers in the evolution ofEchinochloa millets (Poaceae)

Echinochloa (Poaceae) includes two domesticated species,Echinochloa utilis (Japanese barnyard millet) andE. frumentacea (Indian sawa millet) and 20–30 wild species. The two millets are morphologically very variable and overlap in spikelet and inflorescence characteristics. Both species are hexaploids based on x = 9. Cytogenetic studies point to the hexaploid wild speciesE. crusgalli andE. colona as possible progenitors ofE. utilis andE. frumentacea, respectively. The tetraploidE. oryzoides is considered as a possible genome donor to wild and domesticated barnyard millet. Markers from Random Amplified Polymorphic DNA method were used to assess the proposed phylogeny and examine the genetic diversity in both domesticated and wild species. The data were analyzed numerically.Echinochloa utilis andE. frumentacea appear very distinct, but grouped withE. crusgalli andE. colona, respectively. The tetraploidE. oryzoides show strong genetic affinity to theE. utilis—E. crusgalli group. The data are in general agreement with the cytogenetic information; however, some disagreements on the interpretation of some of the cytogenetic information is raised. The variability in DNA markers observed in the domesticated species, particularlyE. frumentacea, points to the feasibility of using RAPD markers in cultivar fingerprinting and breeding programs of these millets.     

Development of eSSR-Markers in Setaria italica and Their Applicability in Studying Genetic Diversity,Cross-Transferability and Comparative Mapping in Millet and Non-Millet Species

Foxtail millet ( Setaria italica L.) is a tractable experimental model crop for studying functional genomics of millets and bioenergy grasses. But the limited availability of genomic resources, particularly expressed sequence-based genic markers is significantly impeding its genetic improvement. Considering this, we attempted to develop EST-derived-SSR (eSSR) markers and utilize them in germplasm characterization, cross-genera transferability and in silico comparative mapping. From 66,027 foxtail millet EST sequences 24,828 non-redundant ESTs were deduced, representing ~16 Mb, which revealed 534 (~2%) eSSRs in 495 SSR containing ESTs at a frequency of 1/30 kb. A total of 447 pp were successfully designed, of which 327 were mapped physically onto nine chromosomes. About 106 selected primer pairs representing the foxtail millet genome showed high-level of cross-genera amplification at an average of ~88% in eight millets and four non-millet species. Broad range of genetic diversity (0.02–0.65) obtained in constructed phylogenetic tree using 40 eSSR markers demonstrated its utility in germplasm characterizations and phylogenetics. Comparative mapping of physically mapped eSSR markers showed considerable proportion of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (~68%), maize (~61%) and rice (~42%) chromosomes. Synteny analysis of eSSRs of foxtail millet, rice, maize and sorghum suggested the nested chromosome fusion frequently observed in grass genomes. Thus, for the first time we had generated large-scale eSSR markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.     

Development and utilization of novel intron length polymorphic markers in foxtail millet (Setaria italica (L.) P. Beauv.)

Introns are noncoding sequences in a gene that are transcribed to precursor mRNA but spliced out during mRNA maturation and are abundant in eukaryotic genomes. The availability of codominant molecular markers and saturated genetic linkage maps have been limited in foxtail millet (Setaria italica (L.) P. Beauv.). Here, we describe the development of 98 novel intron length polymorphic (ILP) markers in foxtail millet using sequence information of the model plant rice. A total of 575 nonredundant expressed sequence tag (EST) sequences were obtained, of which 327 and 248 unique sequences were from dehydration- and salinity-stressed suppression subtractive hybridization libraries, respectively. The BLAST analysis of 98 EST sequences suggests a nearly defined function for about 64% of them, and they were grouped into 11 different functional categories. All 98 ILP primer pairs showed a high level of cross-species amplification in two millets and two nonmillets species ranging from 90% to 100%, with a mean of ～97%. The mean observed heterozygosity and Nei's average gene diversity 0.016 and 0.171, respectively, established the efficiency of the ILP markers for distinguishing the foxtail millet accessions. Based on 26 ILP markers, a reasonable dendrogram of 45 foxtail millet accessions was constructed, demonstrating the utility of ILP markers in germplasm characterizations and genomic relationships in millets and nonmillets species.     

Production of herbicide-resistant transgenic maize plants using electroporation of seed-derived embryos

The improvement of commercial maize lines via biotechnological approaches is limited by the lack of a transformation system that is tissue culture free. In this paper, the development of a genetic transformation system is presented using electroporation for gene delivery and seed-derived embryo as the gene target. Plasmid DNA (pBARGUS), which contained the selectablebar gene for resistance to the herbicide Basta and the screenablegus gene, was delivered into enzymatically wounded mature maize embryos via electroporation. Transformed plants were identified by their ability to grow on a selective medium containing 30 mg/L of phosphinothricin. Southern hybridization, plant resistance to the application of Basta, GUS expression, and segregation analysis indicated that a functionalbar gene had integrated into the maize genome and was inherited in a mendelian fashion by the progeny.     

Applications of biotechnology in eggplant

Eggplant (Solanum melongena L.), an economically important vegetable crop in many countries in Asia and Africa, often has insufficient levels of resistance to biotic and abiotic stresses. Genetic resources of eggplant have been assessed for resistance against its most serious diseases and pests (bacterial and fungal wilts, nematodes and shoot and fruit borer). Attempts at crossing eggplant with its wild relatives resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to respond well in tissue culture, notably plant regeneration, has allowed the application of biotechnology, particularly the exploitation of somaclonal variation, haploidisation, somatic hybridisation and genetic transformation for gene transfer. Somaclonal variation has been used to obtain lines with increased resistance to salt and little leaf disease. Traits of resistance against bacterial and fungal wilts have successfully been introduced into the cultivated eggplant through somatic hybridisation. However, most somatic hybrids were sterile when the parental lines were distantly related. In contrast, the use of close relatives as fusion partners or highly asymmetric fusion resulted in the production of fertile hybrids with resistance traits and a morphology close to the cultivated eggplant, thus avoiding the series of backcrosses necessary for introgression of desired traits into eggplant. As far as molecular markers and genetic engineering are concerned, the information available for eggplant is very scanty. Two genetic linkage maps have been established by using RAPD and RFLP markers. In order to analyse the genetic relationships between eggplant and its relatives, some studies based on AFLP and ctDNA analyses have also been conducted. So far only resistance against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation. However, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently been initiated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improved expression of streptomycin resistance in plants due to a deletion in the streptomycin phosphotransferase coding sequence

Summary Previous studies have shown that a chimeric streptomycin phosphotransferase (SPT) gene can function as a dominant marker for plant cell transformation. The SPT marker previously described by Jones and co-workers has a limited value since it conferred a useful level of resistance only to a fraction (10%) of Nicotiana plumbaginifolia transgenic lines. Expression of resistance was species specific: no such resistant transformants were found in N. tabacum. In this paper we describe an improved SPT construct that utilizes a mutant Tn5 SPT gene. The mutant gene, SPT ^*, encodes a protein with a two amino acid deletion close to its COOH-terminus. In N. tabacum cell culture the efficiency of transformation with the improved streptomycin resistance marker was comparable to kanamycin resistance. When the chimeric SPT ^* gene was introduced linked to a kanamycin resistance gene, streptomycin resistance was expressed in most of the transgenic N. tabacum lines.     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

Controlling Transgene Escape in GM Creeping Bentgrass

Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possible transgene escape to wild and non-transformed species raises ecological and commercial concerns. Male sterility provides an effective way for interrupting gene flow. We have designed and synthesized two chimeric gene constructs consisting of a rice tapetum-specific promoter (TAP) fused to either a ribonuclease gene barnase, or the antisense of a rice tapetum-specific gene rts. Both constructs were linked to the bar gene for selection by resistance to the herbicide glufosinate. Agrobacterium-mediated transformation of creeping bentgrass (cv Penn A-4) with both constructs resulted in herbicide-resistant transgenic plants that were also 100% pollen sterile. Mendelian segregation of herbicide resistance and male sterility was observed in T1 progeny derived from crosses with wild-type plants. Controlled self- and cross-pollination studies showed no gene transfer to non-transgenic plants from male-sterile transgenic plants. Thus, male sterility can serve as an important tool to control transgene escape in bentgrass, facilitating the application of genetic engineering in producing environmentally responsible turfgrass with enhanced traits. It also provides a tool to control gene flow in other perennial species using transgenic technology.     

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.     

谷子茎尖体外遗传转化体系的建立与优化

以谷子(Seteria italica)豫谷一号为实验材料,建立了一套简便、稳定的体外茎尖遗传转化体系。通过根癌农杆菌(Agrobacterium tumefaciens)介导的茎尖转化法,对转化受体采取不同的处理方式,待拟转化株长到三叶期后进行PCR鉴定。探明了草丁膦(Basta)喷施处理用于谷子转基因幼苗筛选的最适...     

鸭疫里默氏杆菌自然转化的发现及机制研究进展

自然转化(natural transformation)是微生物水平基因转移的一种重要机制,其在遗传多样性的产生或修复DNA损伤等方面发挥着重要作用,并且与耐药基因、毒力因子的扩散息息相关。鸭疫里默氏杆菌(Riemerellaanatipestifer,RA)是威克斯菌科中第一个被发现可以发生自然转化的细菌,本文作者利用此特点建立了多种基因编辑的方法,促进了对其遗传多样性和致病机理的研究进程。通过系统性地研究影响鸭疫里默氏杆菌自然转化的因素,鉴定出了参与该菌自然转化的营养物质;通过筛选转座子插入突变体文库,鉴定出了参与该过程的必需基因;最终,在该菌发现了一种新型的自然转化系统。本文结合其他细菌自然转化研究进展,针对以上研究结果进行综述,以期对该菌的自然转化机制有更深入的理解,也为更进一步探明该菌的耐药和毒力基因获得机制提供参考。     

Use of ri-mediated transformation for production of transgenic plants

Summary Agrobacterium rhizogenes-mediated transformation has been used to obtain transgenic plants in 89 different taxa, representing 79 species from 55 genera and 27 families. A diverse range of dicotyledonous plant families is represented, including one Gymnosperm family. In addition to the Ri plasmid, over half these plants have been transformed with foreign genes, including agronomically useful traits. Plants regenerated from hairy roots often show altered plant morphology such as dwarfing, increased rooting, altered flowering, wrinkled leaves and/or increased branching due to rol gene expression. These altered phenotypic features can have potential applications for plant improvement especially in the horticultural industry where such morphological alterations may be desirable. Use of A. rhizogenes and rol gene transformation has tremendous potential for genetic manipulation of plants and has been of particular benefit for improvement of ornamental and woody plants.     

Enzyme diversity in pearl millet (Pennisetum glaucum)

Summary Polymorphism in twelve genes coding for eight enzymes in pearl millet (Pennisetum glaucum (L.) R. Br.): alcohol dehydrogenases (ADH), catalases (CAT), -esterases (EST), glutamate oxaloacetate transaminases (GOT), malate dehydrogenases (MDH), 6-Phosphogluconate dehydrogenases (PGD), phosphoglucoisomerases (PGI) and phosphoglucomutases (PGM), was observed by electrophoresis on 74 cultivated samples and 8 wild samples from West Africa. Six genes: Est A, Adh A, Pgm A, Cat A, Pgi A, Pgd A contain 95% of the total variation. Principal component analyses and discriminant analyses of the 82 samples described by 46 allelic frequencies showed an almost complete separation into 3 groups: wilds, early maturing cultivars and late maturing cultivars. The early group has the highest enzyme diversity, with cultivated millets from Niger showing the most diversity. The high diversity of the early group and its extensive divergence from West-African wild millets suggest, firstly, the existence, elsewhere in Africa of other enzymatically different sources of wild millet, and secondly, the occurrence, prehistorically, of several different domestications. The late group of cultivars has the lowest variability and a relatively low coefficient of differentiation. This relatively homogeneous enzyme structure does not seem to be associated to ecology. A hypothesis is advanced suggesting that West African late-cultivars were derived from a common cultivated early complex. This complex must have been distributed across the Sudanian zone and must have been later sumitted to modifications by limited gene flow with local early maturing cultivars.     

Ornamental Chrysanthemums: Improvement by Biotechnology

The in vitro tissue culture and micropropagation of chrysanthemums, important floricultural (cut-flower) and ornamental (pot and garden) plants, have been well studied. An increase in genetic transformation studies aimed at improving aesthetic and growth characteristics of the plants has been hampered by low transformation efficiencies and genotype dependence of protocols. As a result chrysanthemum regeneration studies have once again emerged as an essential complement of transformation studies. This review highlights the impact that biotechnology has had on the improvement of chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.