Clonal Crops Research Articles

Genetic basis for folk classification of oca (Oxalis tuberosa Molina; Oxalidaceae): implications for research and conservation of clonally propagated crops

Clonally propagated crops exhibit great diversity and are integral components of global and regional food systems. At the same time, little is known about the mechanisms that generate diversity within clonal crop species, and this diversity is increasingly threatened by economic, environmental, and social change. Research addressing the genetic basis for folk classification of clonal crops can address both of these challenges. Here, we carry out such research through a case study of the Andean tuber crop, oca (Oxalis tuberosa Molina). We employ ethnobotanical and molecular genetic methods to assess the congruence in partitioning of 216 oca accessions with respect to 26 folk taxa and with respect to 31 genetic clones. We find that the greatest number of folk taxa (11) correspond to single, unique genetic clones, but we also identify two instances of single folk taxa comprising multiple genetic clones and two instances of multiple folk taxa comprising single, shared genetic clones. We discuss the potential roles of different diversity-generating mechanisms, such as somaclonal variation and sexual reproduction, underlying these varied forms of congruence in order to inspire more directed research on this topic. We also discuss the implications of our findings on in situ and ex situ conservation work, in which practitioners often approximate crop genetic diversity by counting folk taxa. Ultimately, we argue that efforts to understand and conserve clonal crop diversity will be most effective when both folk classification and its genetic basis are considered together.

In vitro technology at the US Potato Genebank

The US Potato Genebank in Sturgeon Bay, Wisconsin, is the national germplasm collection for the world’s most important vegetable crop. It contains about 6,000 accessions of 100 species of tuber-bearing relatives of Solanum tuberosum. The potato of commerce is a clonal crop susceptible to many systemic pathogens, so the genebank routinely uses in vitro clonal maintenance and distribution for named cultivars. In vitro management is also the tool of choice for various breeding and genetic stocks of interest to breeders and researchers. Long-term backup cryopreservation of clones is done at the base collection in Fort Collins, Colorado. In vitro techniques also play an important role in virus elimination from clones. Recently, in vitro propagation has expanded to the temporary safekeeping of meristem propagules in antimicrobial medium during plant collecting expeditions in the southwest USA. The genebank’s mission includes promoting technology that supports expanded use of the germplasm, in particular, finding ways to overcome interspecific hybridization barriers. Thus, in vitro techniques such as pollen viability testing, ploidy manipulation, protoplast fusion, and embryo rescue have contributed technology for major advances in interspecific hybridization, utilization of noncommercial species, and introgression of genes from wild near-relatives. Finally, advancing in vitro technology holds promise as a tool for mass bioassay and selection of seeds, pollen, or somaclones for useful traits.

CRYOPRESERVATION OF CLONAL CROPS: A REVIEW OF KEY PARAMETERS

For a cryopreservation protocol to be successful or to provide optimal results, experimental conditions for each of its successive steps must be optimized. This paper presents some of the parameters, which have to be considered during the establishment of a cryopreservation protocol for clonal crop species. A prerequisite to the development of a cryopreservation protocol for any plant species is the availability of efficient in vitro culture protocols. Relevant in vitro protocols including micropropagation, shoot tip and in some cases meristem culture have been developed for numerous clonal crops. The physiological state of in vitro mother-plants needs to be optimized. In case of temperate species, a cold acclimation period, which triggers cold adaptation mechanisms, is often beneficial. For tropical, cold-sensitive species, this cold acclimation period can be efficiently replaced by a culture period on medium with high sucrose concentration. In order to produce large numbers of homogeneous and actively growing apices, in vitro plantlets can be cut into single node microcuttings, from which shoot tips are excised after a determined period. The composition of the recovery medium for excised shoot tips has to be optimized. Of particular importance is the hormonal composition of the recovery medium. Accessions differing in their in vitro reactivity should be included in all experiments to observe the effect of this parameter on their response to cryopreservation. Among the various techniques available, encapsulation-dehydration and droplet-vitrification are particularly well adapted for cryopreserving shoot tips. The recently developed D-cryoplate and V-cryoplate techniques should also be considered due to their high efficiency and operational simplicity. Various parameters should be taken into consideration for the selection of the optimal technique(s), including notably the type of explants, their sensitivity to cryoprotectants and the environmental conditions. A series of alternative loading and vitrification solutions have been developed recently, which can be efficiently used when the classical solutions prove to be toxic or do not afford the dehydration and desired cryopreservation tolerance. In conclusion, the development of cryopreservation protocols for clonal crops and their large scale application can be expected in a foreseeable future, thanks to the availability of relevant in vitro culture and highly efficient cryopreservation techniques.

CRYOPRESERVATION OF STRAWBERRY GENETIC RESOURCES IN GERMANY

The National German Strawberry Genebank has 369 cultivars and 318 accessions of wild Fragaria species maintained in a field collection at Dresden-Pillnitz. Conservation of clonal crops requires safety duplication. A calculation of the efforts required to establish and maintain a safety backup of the whole collection with in vitro cold storage indicated that it is too labour intensive to maintain the collection in such form. The development of an effective method for cryopreservation is required for a cost effective long-term storage. Four distinct cryopreservation protocols were tested: two protocols using Plant Vitrification Solution 2 (PVS2), encapsulation dehydration and controlled rate cooling. All four methods were equally effective for the cultivars tested, however the Fragaria wild species had significantly better regrowth with the PVS2 vitrification protocol that included 14 d alternating-temperature cold acclimation (16 h at -1°C and 8 h at 22°C). The PVS2 vitrification protocol with cold acclimation will be applied as the standard method for cryopreservation of Fragaria cultivars and wild species. Currently, the Dresden-Pillnitz Institute has 23 different Fragaria accessions in cryopreservation.

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

BackgroundThe recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.).ResultsA modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation.ConclusionsA new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event.

Facilitating the recovery of phenotypically normal transgenic lines in clonal crops: a new strategy illustrated in potato

Transgenic plants frequently exhibit altered phenotypes, unrelated to transgene expression, which are attributed to tissue culture-induced variation and/or insertional mutagenesis. Distinguishing between these possibilities has been difficult in clonal crops such as potato, due to their highly heterozygous background and the resulting inherent phenotypic variability associated with segregation. This study reports the use of transgene integration as a molecular marker to trace the clonal origin of single cells in tissue culture. Following transformation, multiple shoots have been regenerated from cell colonies of potato (Solanum tuberosum L.) and Southern analysis used to confirm their derivation from a single transformed cell. Analysis of phenotypic variation in field trials has demonstrated marked differences between these multiple regeneration events, the origin of which must have occurred after T-DNA insertion, and consequently during the tissue culture phase. This result unequivocally demonstrates that somaclonal variation occurs during tissue culture and independent of transgene insertion. Furthermore, the first shoots recovered do not necessarily exhibit less somaclonal variation, since later regeneration events can give rise to plants that are more phenotypically normal. Therefore, when developing transgenic lines for genetic improvement of clonal crops, multiple shoots should be regenerated and evaluated from each transformation event to facilitate the recovery of phenotypically normal transgenic lines.

Did backcrossing contribute to the origin of hybrid edible bananas?

BackgroundBananas and plantains (Musa spp.) provide a staple food for many millions of people living in the humid tropics. The cultivated varieties (cultivars) are seedless parthenocarpic clones of which the origin remains unclear. Many are believed to be diploid and polyploid hybrids involving the A genome diploid M. acuminata and the B genome M. balbisiana, with the hybrid genomes consisting of a simple combination of the parental ones. Thus the genomic constitution of the diploids has been classified as AB, and that of the triploids as AAB or ABB. However, the morphology of many accessions is biased towards either the A or B phenotype and does not conform to predictions based on these genomic formulae.ScopeOn the basis of published cytotypes (mitochondrial and chloroplast genomes), we speculate here that the hybrid banana genomes are unbalanced with respect to the parental ones, and/or that inter-genome translocation chromosomes are relatively common. We hypothesize that the evolution under domestication of cultivated banana hybrids is more likely to have passed through an intermediate hybrid, which was then involved in a variety of backcrossing events. We present experimental data supporting our hypothesis and we propose a set of experimental approaches to test it, thereby indicating other possibilities for explaining some of the unbalanced genome expressions. Progress in this area would not only throw more light on the origin of one of the most important crops, but provide data of general relevance for the evolution under domestication of many other important clonal crops. At the same time, a complex origin of the cultivated banana hybrids would imply a reconsideration of current breeding strategies.

The evolutionary ecology of clonally propagated domesticated plants

While seed-propagated crops have contributed many evolutionary insights, evolutionary biologists have often neglected clonally propagated crops. We argue that widespread notions about their evolution under domestication are oversimplified, and that they offer rich material for evolutionary studies. The diversity of their wild ancestors, the diverse ecologies of the crop populations themselves, and the intricate mix of selection pressures, acting not only on the parts harvested but also on the parts used by humans to make clonal propagules, result in complex and diverse evolutionary trajectories under domestication. We examine why farmers propagate some plants clonally, and discuss the evolutionary dynamics of sexual reproduction in clonal crops. We explore how their mixed clonal/sexual reproductive systems function, based on the sole example studied in detail, cassava (Manihot esculenta). Biotechnology is now expanding the number of clonal crops, continuing the 10 000-yr-old trend to increase crop yields by propagating elite genotypes. In an era of rapid global change, it is more important than ever to understand how the adaptive potential of clonal crops can be maintained. A key component of strategies for preserving this adaptive potential is the maintenance of mixed clonal/sexual systems, which can be achieved by encouraging and valuing farmer knowledge about the sexual reproductive biology of their clonal crops.

Repeatability Between Two Intermediate Sugarcane Genotype Selection Stages in Florida

Improved yield and disease resistance on sand soils are priorities of the Canal Point (CP) sugarcane (Saccharum spp.) breeding and selection program. Analyses of historical phenotypic data can provide helpful information in guiding selection strategies to meet these priorities. Correlation analysis was used to examine repeatability of phenotypic data used to advance genotypes from an unreplicated single location clonal crop test (stage II) to the subsequent stage (stage III; two replicate, four location clonal crop experiment). Correlations between data for four traits measured in stage II and the corresponding data pooled across soil types for the same genotypes in stage III varied across 23 series of the CP program. Generally, when correlations were statistically significant (P < 0.05), correlation values were low (means; theoretical recoverable sucrose (TRS) r = 0.40, cane yield r = 0.27, and economic index r = 0.23). Similar trends were evident for correlations between data from stage II and stage III on muck soil and stage II and stage III on sand soil across 10 series of the CP program. A 10% reduction in the number of genotypes advanced to stage III over that period would have meant losing only 1 and 13 genotypes that had commercial potential on muck and sand soils, respectively (n = 1278). Correlations between the phenotypic data were significant only for stage III comparisons between TRS and cane yield, which were negatively associated on either soil type. These results indicate that changes in the advancement strategy from stage II are not required as advancing approximately 135 genotypes identifies almost all genotypes with the genetic potential to yield well on muck or sand soils in stage III. Increasing genotypes in stages prior to stage III and changing crossing strategies to improve identification of disease-resistant, high-yielding genotypes for sand soils is recommended.

Unbalanced Bulk of Parents’ Seed does not Cause Significant Drift in Germplasm Regeneration of Two Model Potato (Solanum) Species Populations

Although the potato of commerce, the autotetraploid Solanum tuberosum is a clonal crop, genebank maintenance of wild and cultivated relatives is commonly done as populations of true seeds. In order to help equalize gamete contribution and prevent genetic drift over seed regenerations, an equal number of seeds from each mother plant may be bulked as a source of parents for future regenerations. To determine if this extra sample is beneficial, we selected two S. andigena populations with great variation of seed production among mother plants. Seed production of each mother plant was recorded over 12 standard seed regenerations and genetic indicators were developed in the form of 105 polymorphic RAPD bands. For each polymorphic band, the variation of seed production among mother plants was used to estimate the potential impact on band phenotypic frequency due to an over-all seed bulk. Most bands were fixed or nearly fixed within at least one of the populations, such that the variation in maternal seedset observed would not have put them at signficant risk of being lost in an over-all bulk. For balanced bulks to provide a significant preservation advantage, a band must be rare in all populations and associated with mother plants that produce significantly fewer seeds. Of over 100 loci assessed here, none met these conditions. We therefore conclude that the duplication of documentation and storage space invested in making balanced seed bulks probably return little genetic benefit to the genebank.

Development of Japanese Persimmon Core Collection by Genetic Distance Sampling Based on SSR Markers

ABSTRACTCore collections have been developed in many seed crops, but the corresponding examples in clonal crops were very few. In China there were plenty of Japanese persimmon (Diospyros kaki Thunb.) germplasms. Using 117 Japanese persimmon accessions from a primary core collection, alternative Nei & Li genetic distance sampling methods based on 11 SSR primer pairs have been compared for developing a core collection. The relative success of the different methods was assessed by counting number of alleles, percentage of polymorphic loci, Shannon diversity index and Nei 's gene diversity index. The results showed that 24 candidate core collections were obtained according to 24 different Nei & Li genetic distances. Among 24 core collections, a core collection, containing 15 accessions, proved to be the best one when the value of genetic distance was 0.17. The number of alleles and percentage ofpolymorphic loci of this best core collection were the same as that of primary core collection, and both Shannon diversity index and Nei's gene diversity index were the highest among the 24 core collections.

An experimental approach to simulate transgene pyramiding for the deployment of cry genes to control potato tuber moth (Phthorimaea operculella)

AbstractAlternating the daily feeding of potato tuber moth (Phthorimaea operculella) larvae (PTM) between isogenic pairs of potato plants provides an effective experimental approach to simulate transgene pyramiding in a clonal crop. This involves an experimental design with all six possible pairwise combinations of two transgenic lines expressing different cry genes and the non‐transgenic control. In this manner, we have simulated the pyramiding of pairwise combinations of cry1Ac9, cry9Aa2 and cry1Ba1 genes in potato and evaluated how pairs of these three cry genes interact to influence the growth rate of PTM larvae. The results show that all combinations of the three cry genes were largely consistent with additive impacts on PTM larval growth, although results from the combination of the cry1Ac9 and cry9Aa2 genes were suggestive of slight synergistic effects. Pyramiding the cry1Ac9, cry9Aa2 and cry1Ba1 genes in potato could therefore provide a more effective strategy to control PTM compared to single cry gene transgenic plants.

Plant Genetic Resources in Japan: Platforms and Destinations to Conserve and Utilize Plant Genetic Diversity

Since 1985, the Genebank project has been implemented as a national program to collect, characterize, evaluate, rejuvenate, conserve and use plant, microorganism and animal genetic resources for food and agriculture. NIAS has functioned as a center-bank of the project in cooperation with other agricultural public and private sectors. This national network on genetic resources for food and agriculture currently preserves approximately 233,000 accessions of crop germplasm and wild relatives of about 1,450 species involving 45,000 accessions of clonal crops. Among them 205,000 accessions are conserved as base collection, while 129,000 accessions, as active collection, are distributed for research purposes under the regulation ofMTA between NIAS Genebank and recipients worldwide. We have undertaken international collaboration focusing on exploration for collecting genetic resources under the regulation of MOU or MOA with collaborating parties and on in situ conservation and on farm management of plant genetic resources in several Asian countries. Research highlights on conservation and utilization of plant genetic resources will be discussed in this report.

Alternative selectable markers for potato transformation using minimal T-DNA vectors

Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance.

Effect of NaCl on in vitro propagation of sweet potato (Ipomoea batatas L.).

In vitro propagation protocols offer a better option for production of quality planting materials in a clonal crop such as sweet potato, which is a food crop of versatile uses. Propagation through axillary shoot proliferation and organogenic and embryogenic regeneration were studied in different genotypes of sweet potato. The addition of NaCl enhanced the rate of multiplication as well as yielded hardy somatic embryos. Optimal doses of NaCl in each mode of propagation were different. The hardy somatic embryos produced in NaCl medium could be stored at 8 degrees C with or without a protective alginate covering. High-frequency germination of stored hardy somatic embryos could facilitate the production of artificial seeds. Plantlets produced with the addition of NaCl in regeneration medium were established in vivo at a high frequency (95-100%). Yield and quality of storage roots developed from artificial seed-propagated plants were comparable with those of source plants.

Rare wild plants of potential or current economic importance in Canada — a list of priorities

A priorization system based on dollar value of plant, degree of relationship to economic genotype, and rarity status was applied to rare plants in Canada. This resulted in priorization of 56 taxa, approximately 1% of wild plants of Canada. Most of these taxa are native species, but a few are infraspecific taxa, and some are introduced. Most of the priorized taxa are plants of the Carolinian region of extreme southern Ontario, which is part of the Mixedwood Plains Ecozone. The provinces with the highest numbers were Ontario with 38, British Columbia with 9, Quebec with 8 and Manitoba with 7. Crops, crop relatives and forages accounted for 38 of the taxa. Twelve were plants providing ingredients for commercial drug products or health foods. Nine were sources of wood. Five were plants used in landscape gardening, land reclamation or erosion control. Native berry producing plants, i.e. relatives of clonal crops for which Canada contains significant germplasm on a world scale, were the largest economic group with 18 taxa. Twenty-six of the priorized plants were herbs, 17 were shrubs and 13 were trees. The genera Amelanchier and Linum were the largest taxonomic groups, each with four taxa. The list and related geographic data can be used to orient monitoring and management, to select and acquire material for ex situ protection, and to select and priorize sites for in situ protection. Key words: Germplasm, protection, rare plants, crops, pharmaceuticals, ornamentals, medicinal plants

Family selection in plant breeding

Plant breeding programmes rarely take explicit practical account of the two sources of genetic variance, namely between and within full-sib families, even though existence of these two sources of variation has long been recognised. This paper refers to inbred and clonal crops, not to outbred, seed-propagated species. Theory suggests that the two variances should be of similar size, sometimes very similar. Good comparisons have never been made because variance within families is laborious to estimate. It is proposed that sets of families be assayed as to means in formal trials and only the best few, judged against standards as having potential for superior segregates, should be exploited thoroughly. The calculation as to approximate equality of genetic variances between and within families is important. The vast majority of families should probably be discarded without further ado, and at considerable economy. Sensible decision-making requires an economic component in order to exploit the trade-off between the cost of the initial trial and families discarded without further cost. The object of this paper is a critical review of practical principles, not a general review of a large and diffuse literature.

Diseases of tropical crops: Problems and controls

Summary Tropical crops are diverse as to botanical origins and as to function; they have numerous diseases, some of them very damaging. A few characteristic crops and their diseases and causal agents are listed for purposes of illustration. There are several approaches to control of which the cultural, legal and chemical have some uses; by far the most important is by breeding ‘horizontal resistance’ (HR) which is cheapest and ‘environmentally friendly’. Sugarcane is specifically instanced as the best example in the world of outstandingly successful HR breeding programmes; vast areas of a clonal crop are grown in good health without the use of chemical controls.

A note on the strategy of breeding clonal crops

It is shown that, in breeding a clonal crop for several diverse environments, it is probably best to select populations in those environments and exchange clonal material extensively at an early stage. An example is drawn from sugarcane. There is evidence in this crop that selection is rather inefficient and that superior genotypes may occur at higher frequencies than would usually be thought likely.

Selection in Six Crops of Sugarcane. I. Repeatability of Three Characters

Phenotypic correlation coefficients for three characters among 6 crops of sugarcane (Saccharum sp.) showed that repeatability for stalk diameter among plant crops was somewhat higher than that for stalk number and Brix, which were about equal. Repeatability for stalk number and diameter was about equal between plant crops and first ratoon crops, but Brix was more repeatable among ratoon than among plant crops.Stalk number and Brix in seedling crops and 1.2‐m clonal crops were not significantly correlated with yield in 4.6‐m clonal crops. Stalk diameter in seedling crops and 1.2‐m clonal crops was significantly correlated with yield in 4.6‐m clonal crops and ranged from 0.17 to 0.34 (.01 level of significance). Thus, stalk diameter was a better selection criterion for yield in early stages of selection than was stalk number or Brix.Since Brix and cane yield were not associated, it should be possible to select high‐yielding clones that also have high Brix.