From that research paper:
Materials and Methods
Plant cultivation. Tobacco seeds (Nicotiana tabacum L. K 326)
were germinated in a mixture consisting of 60% (w/w) peat culture
substrate, 20% (w/w) ground maize stalk and 20% (w/w) perlite,
and grown in a seedbed in a naturally illuminated greenhouse for
60 d. Afterwards, the seedlings were washed with tap water to remove
all substrates from the roots, and then transferred to 2.1 L porcelain
pots (one plant per pot) with 1/4 strength aerated nutrient solution.
The solution consisted of (in mM for full strength): 3 NH4NO3;
1 KH2PO4; 2 K2SO4; 2 CaCl2; 2.5 x 10‑1 MgSO4; 2.5 x 10‑2
KCl; 1.25 x 10‑2 H3BO3; 1 x 10‑3 MnSO4; 1 x 10‑3 ZnSO4; 2.5 x
10‑4 CuSO4; 1 x 10‑1 Fe‑EDTA; 2.5 x 10‑4(NH4)
6Mo7O24. Plants
were grown in a growth room with a 14‑h photoperiod. The
photosynthetically active radiation at the surface of the pots was
220–270 mmol m‑2 s
‑1 provided by reflector sunlight metal halide
lamps (Philip Hpiplus, 250W, Belgium).
Treatments and harvest procedures. After 5 d growth in 1/4
strength nutrient solution, the plants were divided into two groups
of 20 plants, each of similar size and development, and full‑strength
nutrient solution with two N levels (1 mM and 6 mM) was supplied.
Nitrogen was provided in the form of NH4NO3. The first harvest
of 5 plants in each group was at 9 d after supply of two N levels.
On the same day, the remaining 15 plants of the two groups were
divided into three sub‑groups and treated as follows: 1) intact plants,
N was replaced by 15N‑labeled NH4NO3 with the same concentra‑
tion (control). 15N was provided as 15NH4
15NO3, produced in
Research Institute of Chemical Industry in Shanghai, China; 2) apex
was excised above the youngest unfolded leaf, number eight from its
base (removing apex), and N was withdrawn from the nutrient solu‑
tion; 3) apex was excised in the same way as in 2), and N was replaced
by 15N‑labeled NH4NO3. The second harvest was at 7 d after the
replacement by 15N‑labeled NH4NO3. There were 5 replicates in
each treatment.
Plant leaves were numbered in ascending order, starting with
the lowest mature leaf, which was designed as leaf 1. Smaller leaves,
which had already senesced, were removed. At the first harvest, the
youngest unfolded leaf was no. 8, and the apical part was incor‑
porated into the upper leaves because of its small size. At harvest,
plants were separated into roots, stems, tops (for the second harvest,
including apex and newly formed leaves after the treatments), the
lower stratum of leaves no. 1‑5 and the upper stratum of leaves no.
6‑8. The lateral buds of the plants whose apex was removed were
removed immediately after their emergence and incorporated into
upper leaves by harvest. All plants parts were dried (70˚C) until
constant dry weight and weighed. They were finely ground (<5 mm)
and used for N and nicotine determinations.
I think we can safely assume that these were extremely immature plants, grown in tiny (2 liter) ceramic pots.
Bob
pubmed.ncbi.nlm.nih.gov
