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Pawpaw in vitro propagation
Giuseppe ZUCCHERELLI Florin STĂNICĂ
University of Agronomic Sciences and Veterinary Medicine
Faculty of Horticulture – Bucureşti
The 4th International Pawpaw Conference - Frankfort -1-3 September 2016
Introduction
– Pawpaw propagation faces few key problems:
- is done mainly by seed, but segregation of the
characters occurs
Introduction
- seedlings have an initial slow growth
- formation of adventitious roots is difficult, no
current propagation by cuttings
Introduction
- clonal multiplication is possible only by grafting
Introduction
- in vitro propagation is an extremely difficult task
- until now, in vitro establishment of shoots, some low
multiplication rate, but no rooting (Finneseth C.L.H. and
Geneve R.L., 2000, Geneve R.L. et al. 2003, Geneve R.L.,
Kester S. and Pomper K. 2007)
Objective
To solve the problem of pawpaw in vitro propagation
by:
- obtaining the in vitro explants rooting,
- obtaining acclimatized plants,
- establish protocols for mass propagation of the best
varieties
Material and method
Place: Vitroplant Italia s.r.l.
Cesena (FC) - ITALY
www.vitroplant.it
Material and method
Biological material:
- Sunflower cv. 15 years old
- Simina
- Potomac
Initial explants:
- apical shoots after bud breaking
Material and method
In vitro establishment:
A- sterilization - ethylic alcohol (90%)
- Vircon (3%) - 30 min.
- sodium hypochlorite (1%) - 25 min.
- three washes with sterile water
B - green house cultivation of the mother plants
C – initial shoot growing in a sterile room
Material and method
Culture media:
- McCown Woody Plant Medium, 1980;
- Murashige & Skoog,1962;
- Quoirin & Lepoivre, 1977;
- Driver & Kuniyuki, 1984.
Hormones balance: BAP 3.0 mg/l, NAA 0.1 mg/l
Growing conditions:
- photoperiod -18 hours,
- light intensity - 4000 lux,
- constant temperature - 22°C.
Material and method
After the third subculture the best media were
modified to obtain stronger plants:
- cytokinines increase with 30%
- auxines reduction with 50%
- sugars increase with 25-30%
“Dark culture” to reduce the emission of polyphenols
Material and method
Rooting – different key points:
- Reduction of the media mineral content;
- Different induction systems based on explants
etiolation and culture on hormones or hormone less
media;
- Use of active charcoals,
and ……many preys!
Material and method
Rooting – media:
- DKW and MS with ½ of minerals
- 3-6-9 mg/l NAA + 3-6-9 mg/l IBA
- 8 days dark root induction treatment
- enriched media with 500 mg/l active charcoal,
Material and method
Acclimatization:
- under automatic environmental controlled
conditions typical for an industrial plant propagation
facility;
Results and discussion
Initiation:
- the normal protocol:
- 50% sterile bud dead explants
- 50% infected explants
Results and discussion
Initiation:
- serious problems with the explants oxidation
- subculture every 20 days to avoid senescence
DKW MS WPM QL
Results and discussion
Multiplication:
- Best results on DKW medium followed by MS
Culture media
Alive
explants
(%)
(3rd subculture)
Shoots
length
(cm)
Healthy
shoots
(%)
McCown Woody Plant -
WPM 5 0.4 0
Murashige&Skoog - MS 25 1.5 10
Quoirin&Lepoivre - QL 15 0.5 5
Driver&Kuniyuki -DKW 35 2.1 15
Results and discussion
Multiplication - Potomac
Results and discussion
Multiplication:
- Best results on DKW medium followed by MS
Results and discussion
Multiplication:
- Best results on modified DKW 980 by
Zuccherelli
Results and discussion
Elongation:
- Best results on modified DKW 980 + 1 mg/l BAP
+0,1 mg/l NAA
Results and discussion
Rooting percentage:
A. Dark induction NAA (mg/l) MS 1/2 DKW 1/2
3 0 0
6 6 5
9 2 1
0 0 0
B. Light root formation + NAA 6 mg/l
IBA (mg/l)
MS 1/2 +
AC 500 mg/l
DKW 1/2 +
AC 500 mg/l
3 45 10
6 85 55
9 72 45
0 0 0
Results and discussion
Rooting: a. dark induction + 6 mg/l NAA,
b. 25 days light root formation + 6 mg/l NAA
Results and discussion
Rooting
Results and discussion
Acclimatization:
Results and discussion
After acclimatization ready for growing
Results and discussion
Acclimatization - Simina
Results and discussion
Acclimatization - Simina
Conclusions
- Species as pawpaw, difficult to be propagated in
vitro, faces serious problems during initiation and
stabilization phase.
- To overcome that, cultivation of mother plants and
initial shoot grow in clean or sterile rooms.
- Short term subcultures and special antioxidant
culture media are needed.
- “Dark culture” is necessary to reduce polyphenols.
Conclusions
- Another important request is, to propagate strong
plants during the multiplication phase able to resist
to the following phases stress: etiolation, high
auxin content, special rooting media etc.
- For species with rooting and growing difficulties
during acclimatization and ex vitro hardening, as
pawpaw, olive, magnolia etc. some special
michorization treatments should be tested
- The presented protocol and results refers to
Sunflower, Simina and Potomac varieties and may
not be applicable to other pawpaw varieties.
Contacts
- Prof. Giuseppe ZUCCHERELLI
Vitroplant Italia s.r.l.
Via Loreto, 170 - 47521 Cesena (FC) - ITALY
Phone: +39 0547 56449
Fax : +39 0547 56711
E-mail: [email protected]
Home page: www.vitroplant.it
- Prof. Florin STĂNICĂ
University of Agronomic Sciences and Veterinary Medicine
Faculty of Horticulture
Bdul Mărăşti, 59 - 011464 Bucureşti - ROMANIA
Phone: +40 722 641795
Fax : +40 21 3182888
E-mail: [email protected]
Home page: www.usamv.ro
Literature cited
Driver J.A., Kuniyuki, Hort. Science , August 1984.
Finneseth, C.L.H., and Geneve, R.L. and Layne, D.R, Establishment of Nord
American PAWPAW Shoots “in vitro” From Mature or Juvenile Explants.
Acta Hort. 520, ISHS 2000.
Geneve R. , Kester S. , Pomper K., Autonomus Shoot Production in PAWPAW,
Propagation of Ornamental Plants, Vol.7 : 51-56. 2007
McCown Woody Plant, Int. Plant. Prop. 30, 421, 1980.
Murashige & Skoog , Physiol. Plant. 15,473, 1962.
Quoirin &Lepoivre, Acta Hort. 78, 437, 1977.
Rupprecht, J.K., et all, 1990. Annonaceus Acetoginins: A Review, J. of Natural
Products, 237-238.
Zuccherelli G. et al. In vitro propagation of fifty olive cultivars. 4th
International
Symposium on Olive Growing, Bari, 2000.
Zuccherelli G., Stănică F. Succesful micropropagation of Asimina triloba
(Dunal). 3rd International Pawpaw Conference. 9-10 September 2011, Frankfort, KY
THANK YOU
FOR YOUR KIND ATTENTION
&
WELCOME
TO ROMANIA AND ITALY!
