Food

Biotechnology Consultation - Note to File BNF 000149

Return to inventory: Completed Consultations on Foods from Genetically Engineered Plant Varieties

See also Biotechnology: Genetically Engineered Plants for Food and Feed and about Submissions on Bioengineered New Plant Varieties


Biotechnology Consultation - Note to File
Biotechnology Notification File No. 149

Date

December 8, 2016

Subject  

EF2-114, Pineapple with altered color containing lycopene and reduced ripening

Keywords

Pineapple, Ananas comosus, carotenoids, PSY, phytoene synthase, bLcy, lycopene β-cyclase, eLcy, lycopene ε-cyclase, RNA interference (RNAi), Citrus unshiu, Nicotiana tabacum, lycopene, OECD Unique Identifier FDP-ØØ114-5, Event EF2-114, Del Monte Fresh Produce Company

Purpose

This document summarizes our evaluation of biotechnology notification file (BNF) No. 000149. The Del Monte Fresh Produce Company (DMFPC) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of genetically engineered pineapple with altered color, transformation event number EF2-114 (EF2-114 pineapple). FDA received it on February 18, 2015. FDA received additional information from DMFPC on January 18, 2016 and August 24, 2016. FDA evaluated the information in DMFPC's submissions to ensure that regulatory and safety issues regarding human food derived from the new EF2-114 pineapple variety have been resolved prior to commercial distribution.

In our evaluation, we considered all information provided by the notifier as well as publicly available information and information in the agency’s files. Here we discuss the outcome of the consultation, but do not intend to restate the information provided in the final consultation in its entirety.

Intended Effects

The intended effects of the modifications in EF2-114 pineapple are to alter the color of the fruit to pink from yellow through accumulation of lycopene in place of β-carotene and to alter flower control. To accomplish this objective, DMFPC introduced genetic material for:

  • One gene (psy) encoding the phytoene synthase (PSY) protein, which is involved in a key step in lycopene synthesis
  • Two RNAi constructs (bLcy and eLcy) intended to suppress endogenous expression of two genes (lycopene β-cyclase and lycopene ε-cyclase) that convert lycopene to other carotenoids
  • One RNAi construct (flACC3ʹ) intended to suppress flowering by suppressing endogenous expression of 1-aminocyclopropane-1-carboxylic acid synthase, the penultimate step in ethylene biosynthesis

Regulatory Considerations

The purposes of this evaluation are to (1) assess whether DMFPC has introduced a substance requiring premarket approval as a food additive into food and (2) to determine whether use of the new plant variety in food raises other regulatory issues under the Federal Food, Drug and Cosmetic Act (FD&C Act). The pineapples would be imported from Costa Rica and would be used solely for human food purposes in the United States.

DMFPC has submitted applications to and obtained authorizations from the Costa Rica Ministry of Agriculture to cultivate EF2-114 pineapple in Costa Rica. DMFPC is consulting with Costa Rican authorities for the necessary food and environmental approvals to commercialize EF2-114 pineapples in Costa Rica.

DMFPC intends to import and distribute EF2-114 pineapples in Canada and is consulting with the Canadian Food Inspection Agency and Health Canada for the necessary food and environmental approvals to do so. 

Genetic Modification and Characterization

Parental Variety

The pineapple variety used to create Event EF2-114 is MD2 a non-genetically engineered conventional variety developed by the Pineapple Research Institute and marketed by DMFPC.

Transformation Plasmid and Method 

Event EF2-114 was developed by co-cultivation of the MD2 variety with two disarmed Agrobacterium tumefaciens GV3101 (pMP90) strains, each containing a binary plasmid vector with the same backbone.

Transfer DNA (T-DNA) from plasmid pHCW.T-7 contains four expression cassettes:

  • The SuRBHra expression cassette, intended to function as a plant selectable marker, which is composed of:
    •  A constitutive fusion promoter EHS-Ubpp, comprising the promoter/5ʹ untranslated region (UTR) of the epoxide hydrolase (EHS) gene and the promoter/5ʹ UTR of the tetrameric ubiquitin (Ubi) gene, both isolated from pineapple
    • SuRBHra, a mutant acetolactate synthase (ALS) gene from tobacco (Nicotiana tabacum[1]
    • A transcription terminator sequence corresponding to the 3ʹ untranslated region of the tobacco ALS gene
  • The BRIp-PSY-Ubpter expression cassette intended to express the phytoene synthase protein, which is composed of:
    • A modified promoter derived from the pineapple BRI gene
    • The tangerine (Citrus unshiu) phytoene synthase (PSY) gene
    • The transcription terminator sequence corresponding to the 3ʹ UTR and flanking region of the pineapple Ubi gene
  • The BRIp-bLcy RNAi-Ubpter expression cassette, intended to suppress expression of the pineapple lycopene β-cyclase protein, which is composed of:
    • A modified promoter derived from the pineapple BRI gene
    • A partial coding sequence of pineapple bLcy in the sense orientation,
    • The intron 2 sequence of the potato (Solanum tuberosum) ST-LSI gene,
    • A partial coding sequence of pineapple bLcy in the anti-sense orientation
    • The transcription terminator sequence corresponding to the 3ʹ UTR and flanking region of the pineapple Ubi gene
  • The BRIp-eLcy RNAi-Ubpter expression cassette, intended to suppress expression of the pineapple lycopene ε-cyclase protein, which is composed of:
    • A modified promoter derived from the pineapple BRI gene
    • A partial coding sequence of pineapple eLcy in the sense orientation
    • The intron 2 sequence of the potato (S. tuberosum) ST-LSI gene
    • A partial coding sequence of pineapple eLcy in the anti-sense orientation
    • The transcription terminator sequence corresponding to the 3ʹ UTR and flanking region of the pineapple Ubi gene 

T-DNA from plasmid pHCWflACC3ʹ-2 contains two expression cassettes:

  • The SuRBHra expression cassette described above, intended as a plant selectable marker
  • The Ubpp-flACC3ʹ RNAi-Upbter expression cassette, intended to suppress expression of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which is composed of:
    • A promoter sequence corresponding to the promoter/5ʹ region of the pineapple Ubi gene
    • A partial coding sequence of pineapple meristem ACS gene flACC3ʹ in the sense orientation
    • The intron 2 sequence of the potato (S. tuberosum) ST-LSI gene
    • A partial coding sequence of pineapple meristem ACS gene flACC3ʹ in the anti-sense orientation
    • The transcription terminator sequence corresponding to the 3ʹ UTR and flanking region of the pineapple Ubi gene
Characteristics and Stability of the Introduced DNA 

Southern Blot Analyses

DMFPC characterized the incorporated DNA using Southern blot hybridization of restriction enzyme digests.

DMFPC conducted analyses to detect any potential vector backbone sequences. Three distinct probes were used to demonstrate that no vector backbone sequences were detectable in the EF2-114 genome.

DMFPC conducted analyses to characterize T-DNA integration. Both plasmids contain the SuRBHra expression cassette adjacent to the right border, permitting use of a single probe to identify right border insertion events associated with both T-DNAs. Eight insertions resulting from either pHCW.T-7 or pHCWflACC3ʹ-2 were identified, including one insertion smaller than the minimum predicted length, suggesting partial integration. A probe unique to the pHCW.T-7 left border identified four insertions, whereas a probe unique to the pHCWflACC3ʹ-2 left border identified a single insertion smaller than the minimum predicted length, suggesting partial integration. The firm concluded that their data was consistent with four complete integrations of the pHCW.T-7 T-DNA, one irregular integration of the pHCWflACC3ʹ-2 T-DNA, and three partial integrations of the pHCWflACC3ʹ-2 right border region.

DMFPC conducted analyses to determine the number of copies of the T-DNA from the two plasmids and their integrity. Because the plasmids shared genetic elements (including elements present endogenously in the pineapple genome), and because the integration events appear complex, these analyses were inconclusive.

DMFPC’s efforts focused on the PSY and bLcy regions of pHCW.T-7 and the SuRBHRa region common to both pHCW.T-7 and pHCWflACC3ʹ-2. The PSY probe produced a single hybridization band of the expected length, with a signal intensity indicating four copies. The bLcy probe produced a band of the predicted size indicating two copies, as well as two additional unexpected bands of varying strength, suggesting the presence of partial or irregular integrations of the bLcy region. The SubHRA probe was used in a test design that produced different fragment sizes for each plasmid. The predicted pHCW.T-7 band was detected with a signal intensity equivalent to four copies. The predicted pHCWflACC3ʹ-2 band was detected with a signal intensity equivalent to two copies. An additional unexpected band was also detected, consistent with a partial or irregular integration. The firm concluded that the data from the copy number analysis was consistent with the integration of four copies of the pHCW.T-7 plasmid, of which two were complete. There was also evidence of one partial integration of the pHCWflACC3ʹ-2 plasmid. The firm considers that Southern blot analysis may not reliably identify copy numbers for complex integration events.

DMFPC conducted analyses to determine whether at least one intact copy of each plasmid was present in EF2-114. Five probes against pHCW.T-7 genetic elements were used to generate data indicating that at least one intact copy of pHCW.T-7 is present in the EF2-114 genome, in addition to rearranged copies of pHCW.T-7, the exact structure of which could not be completely elucidated. Three probes against pHCWflACC3ʹ-2 genetic elements were used in combination with five enzymatic digestion conditions to generate data that supports that no intact copies of pHCWflACC3ʹ-2 are present in EF2-114, and that extensive rearrangements of pHCWflACC3ʹ-2 genetic elements had occurred. The firm notes that hairpin structures like those of some genetic elements in both pHCW.T-7 and pHCWflACC3ʹ-2 have been known to cause rearrangements during integration.

DMFPC conducted analyses to assess the stability of the introduced genetic material in EF2-114 pineapple over four vegetative generations, using a probe against the shared right border region found in both plasmids. The same hybridization pattern was found in all four generations. The firm concluded that this provided evidence of the stability of the introduced genetic material. As a supplementary measure, the firm also assessed the altered color phenotype over six generations and considered it stable.

Flanking Sequence Analysis

DMFPC used thermal asymmetric interlaced (TAIL) PCR to further characterize the right and left border integration events in EF2-114. The firm’s TAIL-PCR analysis identified seven right border flanking sequences and six left border flanking sequences in EF2-114. DMFPC analyzed all potential open reading frames in these flanking sequences and identified no homology with any known allergen or protein toxin.   

Characterization of Introduced Expression Products

EF2-114 pineapple contains introduced genetic material encoding for two protein products (PSY and SuRBHra). Phytoene synthase is a major enzyme in the carotenoid biosynthetic pathway in many plants; the specific PSY in EF2-114 pineapple comes from tangerine, and thus has an extensive history of consumption in food without known concerns. Acetolactate synthase (ALS) catalyzes the conversion of two pyruvate molecules to carbon dioxide and an acetolactate molecule, necessary for production of valine, leucine, and isoleucine and is present in all plants. The specific ALS used in EF2-114 is derived from tobacco and is over 90% identical in sequence to other Solanaceous ALS enzymes with an extensive history of consumption in food (tomato, potato, pepper). Replacement of two amino acids in SuRBHra yields an ALS variant that confers tolerance to sulfonylurea herbicides. DMFPC notes that similarly modified ALS enzymes from corn and soybean have been traits in new plants varieties that were the subjects of earlier submissions to FDA and that no concerns have been identified with respect to these proteins. 

DMFPC reports that attempts to isolate PSY and SuRBHra from pineapple tissue were unsuccessful due to technical challenges. (The proteins also proved challenging to express in and extract from genetically engineered Escherichia coli.) An attempt to characterize tissue expression of the RNA transcripts via Northern blot was also unsuccessful. The firm ultimately used quantitative reverse transcriptase-PCR to quantify RNA expression of the introduced genetic material coding for PSY and SuRBHra. PSY transcripts were below the level of detection. SuRBHra transcripts were detected at low levels relative to other endogenous proteins. The firm concludes that endogenous PSY expression and phytoene production remained sufficient to achieve the desired lycopene phenotype. 

DMFPC also used qRT-PCR to quantify the effect of the introduced genetic material encoding for bLcy, eLcy, and flACC RNAi. Endogenous transcripts of both bLcy and eLcy were significantly reduced in EF2-114 compared to MD2, consistent with the observed increase in lycopene levels. The firm could not detect flACC transcripts in either MD2 or EF2-114 plants, and thus could not assess the impact of the introduced flACC RNAi construct. However, no flower control phenotype was observed in field trials, as would be expected from the absence of a complete integration of pHCWflACC3ʹ-2 T-DNA supported by the genetic analysis. 

Bioinformatics studies using standard methods revealed no significant homology to known protein toxins or allergens for either PSY or SuBHra.

Food & Feed Use

Pineapple (Ananas comosus var comosus) is a herbaceous perennial crop native to the tropical Americas. Several cultivars are grown commercially in tropical and subtropical countries around the world. Commercial propagation is exclusively vegetative. Pineapple is cultivated for its fruit, which is consumed as food in both fresh and processed forms. The fruit is a good source of vitamin C and potassium.

Fresh, dry, and ensiled pineapple wastes are sometimes used as animal feed. DMFPC does not anticipate that any meaningful amount of pineapple byproducts derived from Event EF2-114 pineapple will be used in animal feeds in the United States. The Center for Veterinary Medicine does not need to conduct an in-depth review of the scientific data provided in the notification because the pineapple variety will not be grown in the United States and pineapples and pineapple by-products are not significant ingredients in animal food in the United States[2].

Composition

Scope of Analysis

DMFPC measured levels of key nutrients[3] in EF2-114 pineapple and the non-genetically engineered parental variety, which is also the predominant pineapple variety consumed in North America MD2 (control). DMFPC also compared the mean values obtained for EF2-114 pineapple to levels reported in scientific literature for pineapple varieties that have a history of safe consumption.

Study Design - Compositional Analyses

DMFPC grew EF2-114 pineapple and the control under identical climactic conditions and cultural practices at a single site in Buenos Aires, Costa Rica; the planting date for EF2-114 and control was May 2011, and the pineapples were harvested in October 2013. DMFPC used a randomized block design with three unequal replicates, consisting of a total of 2,541 EF2-114 and 145 control plants. Fruits for compositional analyses were harvested at the same ripening stage and size. 18 EF2-114 and control pineapples each were randomly selected for compositional analyses. DMFPC statistically compared compositional values for components from EF2-114 pineapple with those from the control using a Student t test. Differences between EF2-114 pineapple and the control were considered statistically significant based on P< 0.01.

Results of analyses:

DMFPC evaluated 19 key components in EF2-114 and control pineapple; these components were chosen based on their importance to fruit quality, flavor, nutrition, and the intended effect of the event. DMFPC reported values for moisture, proximates (crude protein, crude fat, carbohydrates by calculation, and ash), total dietary fiber, sugars (sucrose, fructose, and glucose), amino acids (leucine, isoleucine, methionine, and valine), potassium, carotenoids (alpha-carotene, beta-carotene, lycopene, lutein), and vitamin C, on a percent fresh weight basis. DMFPC observed a statistically significant difference (P<0.001) between the mean values of potassium levels in EF2-114 pineapple and the control. DMFPC attributed this observed difference to environmental and post-harvest factors; however, DMFPC also noted that the mean values for potassium were within the reported literature values for pineapple varieties. DMFPC also observed small statistically significant differences (P<0.01) between the mean values for ash, carbohydrates by calculation, moisture, sucrose, vitamin C, and leucine in EF2-114 pineapple and the control; however, the mean values obtained were within the reported ranges of values for pineapple varieties in the scientific literature.

Intended Effect

DMFPC found a statistically significant difference (P<0.001) between the mean values of lycopene and beta-carotene, in EF2-114 pineapple and the control; this was expected. The mean level of lycopene in EF2-114 pineapple was 21.3 ppm. The levels of lycopene in EF2-114 pineapple are similar to that present in commonly consumed fruits such as tomato, watermelon, and grapefruit. The mean levels of beta carotene were observed to be 5.34μg/100g in EF2-114 pineapple, compared to 16.86 μg/100g in the control[4].

Summary of Compositional Analyses

Based on the results obtained, DMFPC concludes that EF2-114 pineapple is compositionally and nutritionally comparable to conventional pineapple varieties except for the intended effect and is as safe as conventional pineapple varieties for its intended uses in human food.  

Food Labeling Considerations

It is a producer’s or distributor’s responsibility to ensure that labeling of the foods it markets meets applicable legal requirements. DMFPC stated that it intends to identify the food as “Extra Sweet Pink Flesh Pineapple.” CFSAN’s Office of Nutrition and Food Labeling, Food Labeling and Standards Staff (ONFL/FLSS) agrees that this name is an accurate description of the EF2-114 pineapple. Companies marketing Event EF2-114 pineapples are advised to consult with ONFL/FLSS to discuss any required or voluntary labeling including statements relating to attributes of these pineapples or any other type of claim.

On July 29, 2016, the National Bioengineered Food Disclosure Law (Public Law 114-216) charged the United States Department of Agriculture’s Agricultural Marketing Service with developing a national mandatory system for disclosing the presence of bioengineered material in food. Producers, distributors, and marketers of EF2-114 pineapple are responsible for following the requirements issued by USDA relevant to the labeling of their products.

Conclusion

FDA evaluated DMFPC’s submission to determine whether the developer’s genetically engineered pineapple variety raises any safety or regulatory issues with respect to the intended modification or with respect to its use in food for humans. We did not evaluate use in animal feed at this time given the information provided by DMFPC. Based on the information provided by the company and other information available to the agency, FDA did not identify any safety or regulatory issues under the FD&C Act that would require further evaluation at this time.

DMFPC has concluded that the human foods derived from EF2-114 pineapple are as safe as and with the exception of altered color (from lycopene) are not materially different in composition or any other relevant parameter from other pineapple varieties now grown, marketed, and consumed. At this time, based on DMFPC’s data and information, the agency considers DMFPC’s consultation on EF2-114 pineapple for use in human food to be complete. 

Jeremiah M. Fasano, Ph.D.


[1] The protein encoded by SuRBHra is resistant to the herbicide chlorsulfuron.

[2] For details, see R. Christensen: Memorandum to File BNF 000149.

[3] DMFPC states that there are no anti-nutrients reported in the published literature in ripened pineapple fruit.

[4] Pineapples are not a major source of beta-carotene.

Page Last Updated: 01/04/2017
Note: If you need help accessing information in different file formats, see Instructions for Downloading Viewers and Players.
Language Assistance Available: Español | 繁體中文 | Tiếng Việt | 한국어 | Tagalog | Русский | العربية | Kreyòl Ayisyen | Français | Polski | Português | Italiano | Deutsch | 日本語 | فارسی | English