Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation.
© Acciarri et al; licensee BioMed Central Ltd. 2002
Received: 20 December 2001
Accepted: 04 April 2002
Published: 04 April 2002
Parthenocarpy, or fruit development in the absence of fertilization, has been genetically engineered in eggplant and in other horticultural species by using the DefH9-iaaM gene. The iaaM gene codes for tryptophan monoxygenase and confers auxin synthesis, while the DefH9 controlling regions drive expression of the gene specifically in the ovules and placenta. A previous greenhouse trial for winter production of genetically engineered (GM) parthenocarpic eggplants demonstrated a significant increase (an average of 33% increase) in fruit production concomitant with a reduction in cultivation costs.
GM parthenocarpic eggplants have been evaluated in three field trials. Two greenhouse spring trials have shown that these plants outyielded the corresponding untransformed genotypes, while a summer trial has shown that improved fruit productivity in GM eggplants can also be achieved in open field cultivation. Since the fruits were always seedless, the quality of GM eggplant fruits was improved as well. RT-PCR analysis demonstrated that the DefH9-iaaM gene is expressed during late stages of fruit development.
The DefH9-iaaM parthenocarpic gene is a biotechnological tool that enhances the agronomic value of all eggplant genotypes tested. The main advantages of DefH9-iaaM eggplants are: i) improved fruit productivity (at least 30–35%) under both greenhouse and open field cultivation; ii) production of good quality (marketable) fruits during different types of cultivation; iii) seedless fruit with improved quality. Such advantages have been achieved without the use of either male or female sterility genes.
Fruit-set and growth of several horticultural plants are negatively affected by adverse environmental conditions. In general, sub and/or supra-optimal temperatures negatively affect reproductive processes and therefore curtail fruit production [1, 2]. Under greenhouse cultivation, low temperature, insufficient light intensity, and/or high humidity drastically reduce fruit productivity and quality in eggplant and other species. Moreover, environmental conditions often met in open field cultivation such as drought and high temperatures have a negative effect on fruit productivity and quality in eggplant and other species (e.g. tomato).
Parthenocarpic fruit development (i.e. fruit-set and growth without fertilization) can significantly aid in the resolution of the aforementioned problems. Parthenocarpy can be triggered by exogenous factors, such as plant growth regulators, or it can be achieved by genetic factors. Genes causing parthenocarpic development have been identified in several plant species [3–5], and parthenocarpic eggplant varieties (e.g. Talina, Galine) have been introduced in the production process (e.g. protected cultivation). However, during winter cultivation of eggplant varieties in unheated greenhouses in the Mediterranean area, the negative effect of suboptimal environmental conditions on fruit production is usually counteracted by treating flower buds with plant growth regulators. Phytohormonal treatments make the production process more expensive due to the cost of both chemicals and labor.
In principle, the genetic engineering of plants allows one to confer a trait of interest to different species and within a species to all the varieties of interest. To confer parthenocarpic fruit development, a chimeric gene has been constructed . Specifically, the DefH9-iaaM gene contains the coding region of the iaaM gene from Pseudomonas syringae pv. savastanoi under the control of the placenta and ovule-specific promoter from the DefH9 gene from Anthirrinum majus. The iaaM gene codes for a tryptophan monoxygenase, which produces indolacetamide that in turn, is either chemically or enzymatically converted to the auxin indole-3-acetic acid. To date, the DefH9-iaaM chimeric gene has been shown to cause parthenocarpic fruit development in tobacco , eggplant , tomato [7, 8], strawberry and raspberry .
We have previously shown that DefH9-iaaM eggplants outperform control plants during protected winter cultivation by an average of 33% . The present manuscript presents data on the agronomic performance of DefH9-iaaM eggplant hybrids during spring and summer cultivation. Seed-derived F1 plants perfectly reflects the real agronomical situation of eggplant production. In addition, they have allowed to demonstrate that the transgene is active after meiosis and give satisfactory results in the hemizygous state. Different types of eggplant hybrids have been evaluated during springtime in unheated greenhouses in two different locations. To evaluate GM parthenocarpic eggplants under optimal environmental conditions, a single trial has been carried out using two different genotypes under standard open field cultivation during summertime. Furthermore, we demonstrate that the DefH9-iaaM gene is expressed during late stages of fruit development.
Results and discussion
Greenhouse spring production
Eggplant production of parthenocarpic hybrids and their respective controls at springtime.
Fruit weight (g)
Fruit weight (g)
The increased productivity of GM hybrids characterised both the early spring production (i.e. the first four harvests) and the whole spring production cycle (i.e. sixteen harvests). During the whole spring production cycle, the hybrids P1 and P2 gave an average yield that was 46% higher with respect to the corresponding control C1 (Table 1). The hybrid P5 gave a 37% higher yield with respect to its control C2. The average total number of fruits produced per plant in both locations was similar in all the hybrids (8–9 fruits/plant). However, the higher average weight of the GM fruits led to a higher total yield of transgenic hybrids with respect to their controls. When considering the whole spring cultivation cycle, the parthenocarpic cultivar Talina gave a total production that was not significantly different from either of the three GM hybrids (Table 1).
Open field (summer) production
Eggplant production of parthenocarpic hybrids and their respective controls at summertime.
Fruit weight (g)
Fruit weight (g)
Although the environmental conditions were optimal and consequently did not affect negatively fruit-set, the DefH9-iaaM gene caused both faster development and growth of the fruits as indicated by the increased early-summer production (the first three harvests). In this regard, it is worthwhile to stress that expression of the DefH9-iaaM gene takes place in the placenta and ovules before pollen development. As a consequence, in GM parthenocarpic plants fruits are seedless and fruit development initiates well before non-GM controls .
In all trials we have never used homozygous lines because growers mostly employ F1 hybrids. The use of hemizygous primary transformants as pollinator plants allowed us to obtain in rather short time, by in vivo selection for kanamycin resistance, F1 plants transgenic for the DefH9-iaaM gene. Young, healthy and vigorously growing plants did not produce seeds. However, it is possible to obtain seeds from aged DefH9-iaaM transgenic plants both by selfing and crossing. By exploiting the delayed female fertility we have produced the homozygous plants needed as male parents for rapid seed multiplication of F1 eggplant hybrids. Therefore, the female sterility of young and mature plants is not an insuperable hindrance for mass propagation and commercial fruit production.
Expression of the DefH9-iaaMgene takes place during both flower and fruit development in transgenic parthenocarpic eggplants
Treatment with auxin often causes parthenocarpic development in several plant species [for review, see: ]. However, in some species and/or varieties, to efficiently sustain fruit growth, the hormonal treatment of the flowers must be repeated . The finding that DefH9-iaaM mRNA is also present during later stages of fruit development is consistent with the interpretation that in DefH9-iaaM parthenocarpic plants, the placenta, the ovules, and the tissues derived therefrom are a source of auxin during the whole growth of the fruit. As a consequence, they efficiently sustain fruit growth.
The data hereby presented show the positive influence of the DefH9-iaaM parthenocarpic gene on eggplant productivity under both greenhouse (spring) and open field (summer) cultivation. Taking into account the data previously obtained under winter greenhouse cultivation , we conservatively estimate that the overall increase in productivity is at least 30–35%. The increase in productivity of DefH9-iaaM eggplants is mainly due to a drastically improved fruit-set under sub-optimal temperatures and to an enhanced fruit growth and weight. Fruit quality is also improved because the fruits are seedless and do not show a placental cavity. The qualitative improvement of DefH9-iaaM eggplant fruits is interesting both for the fresh market and for the processing industry. During early spring greenhouse production, DefH9-iaaM parthenocarpic hybrids always gave fruits with an average weight suitable for fresh market commercialization, while untransformed hybrids, under sub-optimal conditions, rarely produced commercial fruits. Thus, the DefH9-iaaM gene quantitatively and qualitatively improves eggplant production under both greenhouse and open field cultivation. In all genotypes tested the DefH9-iaaM gene had a very positive effect on production and quality parameters. Such findings are of paramount importance as the hybrids tested have the same genetic background that the relative controls, except for the presence of the DefH9-iaaM gene. The DefH9-iaaM gene, already known to be expressed in the placenta and ovules during early phases of flower development, is expressed also in mature fruits, most likely in tissues derived from the ovules.
From an economical standpoint, the main advantages conferred to eggplant by the DefH9-iaaM gene are: i) production of marketable fruits under environmental conditions adverse for fruit-set and growth; ii) reduction of cultivation costs (energy, phytohormones and labor) necessary for off-season and open field eggplant cultivation; and iii) enhancement of fruit quality. Last but not least, contrary to conventional wisdom, these advantages have been achieved without the use of either male or female sterility genes.
Materials and methods
Greenhouse spring cultivation
Open field (summer) cultivation
The open field trial was carried out under open field conditions at Monsampolo del Tronto (approval of the Italian Ministry of Health B/IT/99/21). Two transgenic parthenocarpic hybrids were tested: the hybrid P1 (Tal1/1 × DR2 iaaM #34-1) with elongated fruits and the hybrid P10 (UGA × Tal1/1 iaaM #1-1) with sub-oval fruits were compared to their homologous non-transgenic controls C1 (DR2 × Tal1/1) and C10 (UGA × Tal1/1), respectively. The UGA line has oval dark purplish fruits and it has been provided by Dr. S.C. Phatak. A complete randomized block design with the hybrids replicated four times was adopted. Each experimental plot measured 11.7 m2 and contained 30 plants in a double row. Transplanting was performed on May 10th.
Early spring production consisted of the first four harvests (i.e. 4 out of 16 harvests during the whole production cycle), while early summer production, whose cultivation cycle consists of ten harvests, corresponds to the first three harvests. For all trials the number and weight of fruits were recorded. In addition, fruit sample for each harvest and replication was cut to check for the presence of seeds. Data were computed for the early harvesting time and for the whole harvesting season. Analysis of variance was performed according to a randomized complete block design. Duncan's Multiple Range Test (P = 0.05) was used for means separations.
Plant DNA isolation and Southern blot analysis
High-molecular-weight DNA was isolated from the young leaves of transgenic and untransformed eggplants by using Plant DNAzol (Invitrogen), according to the manufacturer's instructions. Ten μg of DNA was digested overnight with 80 units of KpnI (Promega) in a volume of 500 μl, separated on a 0.7% agarose gel and transferred to Hybond N (Amersham Pharmacia Biotech). A 1350 bp fragment of the DefH9 gene was used as a radiolabeled probe. The membrane was hybridized overnight in 5X SSC/50% formamide (Sigma) at 42°C and washed two times for 15 min. in 2 × SSC/0.1% SDS, and two times for 15 min. in 0.1 × SSC/0.1% SDS at 42°C. Signals were detected using Kodak X-OMAT AR5 film (Sigma).
Semiquantitative (competitive) PCR analysis was carried out for 38 cycles (annealing temperature 63°C) using as template cDNA (8 ng) obtained by priming poly(A)+ mRNA with an iaaM specific primer (5'-AATAGCTGCCTATGCCTCCCGTCAT-3'). The mRNA was extracted from either young flower buds (5,8,11 mm) or eggplant fruits (placental tissue from fruits either 40 or 280 mm long). As an internal standard, 0.5 fg of a 600 bp long DefH9 cDNA fragment was used in the PCR assay. To amplify the 161 bp long amplicon an iaaM specific primer (5'-GGGTGAATTAAAATGGTCATACAT-3') and a DefH9 specific primer (5'-CTTTGGAACTCGTGTTGAGCTCTCA-3') were used. For the internal standard, a 3' primer (5'-TGAGCATTGATCTCCTGAGTGGTGT-3') together with the DefH9 specific primer were used to produce the 351 bp long amplicon. The PCR assays were performed with a thermostable DNA polymerase mixture (Expand High Fidelity PCR system, Roche) in presence of 3 μCi of 32P dCTP. The intensity of the bands was quantified by using an Instant Imager (Packard, Meriden, CT).
This research was partially supported by the Consiglio Nazionale delle Ricerce (Progetto Finalizzato Biotecnologie II) and by the Ministero Politiche Agricole e Forestali Progetto "Biotecnologie vegetali". We thank Prof. Phatak, University of Georgia (USA) for the UGA line.
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