1. CONCLUSIONS AND FUTURE WORK
ABSTRACT
METHODOLOGY AND RESULTS
This research is financed by the USDA‐Hatch project H‐457
Martha C. Giraldo‐Zapata1 and Luis A. Sánchez2
1Assistant Professor And 2Research Associate, Agricultural Experiment Station at Corozal. Agroenvironmental Sciences Department,
University of Puerto Rico, Mayagüez, PR 00681.P.O. Box 9000. Correspondent author: martha.giraldo@upr.edu
OBJECTIVES
Apio (Arracacia xanthorrhiza Bancroft) is produced in the north‐central region of the Island.
Especially at the municipalities of Barranquitas and Orocovis. This starchy crop is vegetatively
propagated and contains the finest starch from all tuber crops. The breakout of a disease called
“apio corm rot” decrease dramatically the quality and quantity of the farmer's propagation
material in this region, diminishing their availability for the next planting season. Currently,
propagation material from this area has a germination rate between 7.5 to 45%. Plant tissue
culture or micropropagation could be used to maintain, grow and increase the availability of
disease free propagation material towards recovery the apio production at this region. There is
a lack of studies that describes a complete protocol for apio micropropagation from
establishment to hardening. Our research at the Agricultural Experiment Station in Corozal
have being focused on the development of a complete protocol for the apio micropropagation
from establishment, multiplication, rooting and hardening. This presentation will describe our
advances in the initial protocols before hardening at the greenhouse. We were able to obtain a
contamination free establishment phase, a multiplication rate around 1:3 and a 95% of rooting.
• Test different culture media for multiplication of Apio.
• Establishment of a micropropagation protocol for Apio.
• Produce enough disease‐free propagation material for the apio farmers.
Explant or Shoot Sterilization
• The selected shoots were washed with a soap solution and running
water for 30 minutes.
• Then, selected shoots containing the apical meristem were reduced into
8 mm length and dipped into 95% (v/v) Ethanol per 15 seconds,
followed by 1% (v/v) Sodium Hypochlorite with 0.1% (v/v) tween 20 for
20 minutes, and washed three times with sterile water.
• A second reduction from 2 to 4 mm length of the shoots are performed
to transferred them into the culture medium.
Plant Material
• The plant material was collected at
Barranquitas, Orocovis and Jayuya.
• From adult plants we select the bigger
shoots produced on the crown of the
main rootstock.
Establishment
The nutrient media, Murashige and Skoog (MS), without hormones, was
inoculated with the sterile shoots and incubated in a culture room where
light and temperature are controlled. The temperature was set between
25‐26°C and a light intensity of 4,000 Lux for 12 hours. Regenerated shoots
were transferred into a fresh culture medium every 3 weeks and
maintained at the same conditions for 2 cycles.
Multiplication
The shoots were transferred on MS media supplemented using 3% (w/v)
sucrose, 0.1% (v/v) Plant Preservative Mixture™ (PPM) at a pH 5.80 and
0.2 % (w/v) Phyto agar. The combination of plant growth regulators (PGR)
was selected from initial experiments comparing a range and
combinations of metaTopolin (mT) ( 2.07, 4.14, 8.28, and 16.56 μM/L) and
1‐naphthaleneacetic acid (NAA) (0.54 μM/L). Medium without PGR were
used as control. Multiple new shoots formed were divided and transferred
into fresh culture medium every 3 weeks and maintained at the same
growth conditions as in the establishment phase for 2‐3 cycles.
METHODOLOGY AND RESULTS
Rooting
Induction of roots on the multiplied shoots was initiated testing two
different media conditions: I.) MS medium, supplemented with 3 %
sucrose , 0.1% (v/v) PPM, 2.07 μM/L metaTopolin, 39.5 μM/L
Phloroglucinol (PG); and II.) Half‐strength MS medium containing 2%
sucrose , 0.1% (v/v) PPM, 39.5 μM/L Phloroglucinol, both with pH 5.80
and 0.2% (w/v) Phyto agar.
Hardening
Vigorous plantlets with properly developed roots were rinsed with tap
water to remove the medium and transferred into sterilized soil mixture
2:1 (v/v) Promix:Sand. Light, temperature and humidity conditions need to
be tested. The plants require gradual acclimatization to the in vivo
environmental conditions in the greenhouse for 30 days. The success rate
of micropropagation depends on the survival of the plantlets at the
greenhouse. This phase still in development.
I. II. II.
Newly Transplanted Two months at the greenhouse
• The most effective protocol for multiplication contains MS culture medium
with NAA (0.54 μM/L) and mT (2.07 μM/L) with a multiplication rate from
3.5 to 4 new shoots in three weeks.
• For rooting the highest rooting frequency (95%) was obtained with a culture
medium half‐strength MS containing 2% sucrose and 39.5 μM/L
Phloroglucinol.
• Research for hardening and improving the survival of the plantlets at the
greenhouse still in progress.
PROTOCOL ESTABLISHMENT FOR APIO (Arracacia xanthorrhiza Bancroft)
PROPAGATION BY TISSUE CULTURE IN PUERTO RICO.