Evaluation of plant seed DNA and botanical evidence for potential forensic applications

Abstract

Seeds, the reproductive organs of plants, are common as trace evidence from crime scenes. Seed evidence could be grouped into several categories based on the types of crimes they are associated with, including child abuse, homicides and drugs. Most commonly, seeds are examined microscopically and identified to the plant species level to show a linkage between persons and places. More recently, forensic researchers have evaluated the potential for extracting and typing DNA from seeds to further individualize the samples. As a model system, tomato seeds were examined microscopically after different cooking treatments and assessed for the potential to DNA type seeds for variety identification. A sufficient quantity and quality of DNA were recovered from uncooked, digested and undigested tomato seeds for amplified fragment length polymorphism (AFLP) analysis; however, any form of cooking destroyed the seed DNA. A simple microscopic analysis was able to distinguish between a cooked tomato seed versus an uncooked seed. This article is intended to provide an overview of case examples and current techniques for the forensic examination of seeds as plant-derived evidence.

Keywords: AFLP; DNA extraction; Forensic sciences; STR; crime scene; forensic botany; non-human evidence; tomato seed.

Figure 1.

Five cultivars of whole tomatoes (letter means color, diameter; (A) red, 3.5 cm²; (B) red, 8.5 cm × 5.4 cm; (C) yellow, 7.0 cm²; (D) red, 7.5 cm²; (E) red, 9.0 cm × 7.7 cm).

Figure 2.

Five cultivars of tomato seeds. It is hard to distinguish different tomato cultivars from seed’s morphological appearance (designated A–E).

Figure 3.

High-quality total genomic DNA could be recovered from dissected embryos (cut in half) and intact tomato seeds (embryo plus seed coat, cut in half) from two of the five tomatoes. Lanes 1–6 are DNA quantitative standards, human K562 (12.5, 25, 50, 100, 200, 400 ng, respectively); lane 7 is half an embryo from tomato 1; lane 8 is half a seed coat from tomato 1; lane 9 is half a seed (embryo + seed coat combined) from tomato 1; lane 10 is half a seed (embryo + seed coat combined) from tomato 2; lane 11 is half a seed coat from tomato 2; lane 12 is half an embryo from tomato 2; lane 13 is an intact seed from tomato 3; lanes 14–16 are standard size markers 100 bp, 1 Kb, and Lambda DNA-Hind III ladder, respectively (New England Biolabs, Beverly, MA, USA). In all cases, the recovered DNA from the tomato seeds or dissected seed components was approximately 12.5 ng/10 μL or less.

Figure 4.

High-quality total genomic DNA was recoverable from a single fresh seed and single fresh tomato digested seed. This DNA was of sufficient PCR quality as tested by the AFLP method. Lanes 1–6 are DNA quantitative standards, human K562 (12.5, 25, 50, 100, 200, 400 ng, respectively) ; lanes 7–10 are single intact fresh tomato seeds 1–4 from one tomato type; lane 11 is from a single, fresh digested tomato seed; lane 12 is a negative control; lanes 13–15 are standard size markers 100 bp, 1 Kb and Lambda DNA-Hind III ladder, respectively (New England Biolabs, Beverly, MA, USA).

Figure 5.

The suggested improvement in tube design may increase single seed DNA yields with the Mixer Mill machine seed preparation method. The cap design is inverted to avoid the space where the seed can become caught between the cap and the tube wall and would result in better tissue disruption and increased DNA yields.

Figure 6.

(A) DNA test results of nine seeds from commercial tomato products. Lanes 1–3 are quantitation standards, K562 (50, 25, 12.5 ng, respectively) ; lane 4 is a negative control; lanes 5–9 are DNA extracted from seeds of five canned tomatoes; lanes 10–13 are DNA extracted from seeds of four brands of tomato spaghetti sauces; lane 14 is a DNA extracted from fresh seed; lanes 15–16 are standard size markers, 1 Kb, and 100 bp ladder, respectively (New England Biolabs, Beverly, MA, USA). (B) DNA extracted from 1 mL of tomato sauce and fresh tomato juice. Lanes 1–4 are quantitative standards, K562 (100, 50, 25, 12.5 ng, respectively); lane 5 is a negative control; lanes 6–7 are DNA extracted from two types of canned tomatoes; lanes 8–13 are DNA extracted from six brands of tomato spaghetti sauces; lane 14 is a DNA extracted from fresh tomato tissue; lanes 15–16 are standard size markers 1 Kb and 100 bp ladder, respectively (New England Biolabs). (C) Photomicrographs of the seed surface morphology of nine brands of commercial tomato products and one fresh tomato seed.

Figure 7.

(A) DNA test results from 10 different cooked conditions. Lanes 1–3 are quantitative standards, K562 (50, 25, 12.5 ng); lane 4 is a negative control; lanes 5–14 are tomato seeds that were boiled for 1, 3, 5, 10, 15, 20, 30 s and 1, 3, 5 min; lanes 15–16 are standard size markers 1 Kb and 100 bp ladder (New England Biolabs, Beverly, MA, USA). (B) DNA test results from 10 different cooked conditions. Lanes 1–3 are quantitative standards, K562 (50, 25, 12.5 ng); lanes 4–9 are tomato seeds that were boiled for 10, 20, 30 min and 1, 2, 3 h; lane 10 is a pan-fried tomato seed that was cooked above 100 °C for 3 min; lanes 11–13 are tomato seeds that were cooked at 218 °C for 10, 20 or 30 min); lane 14 is DNA from fresh seed; lanes 15–16 are standard size markers 1 Kb and 100 bp ladder (New England Biolabs). (C) Photomicrographs of the seed surface morphology from seeds cooked at 10 different cooking times (from 30 s to 3 h) at 100 °C and one fresh tomato seed.

Figure 8.

Photomicrographs of the seed surface morphology of fresh and cooked (pan-fried and baked) seeds.