Pawpaw in vitro propagation

Giuseppe ZUCCHERELLI Florin STĂNICĂ

flstanica@yahoo.co.uk

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: contact@vitroplant.it

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: florin.stanica@usamv.ro

Home page: www.usamv.ro

mailto:contact@vitroplant.ithttp://www.vitroplant.it/mailto:contact@vitroplant.ithttp://www.vitroplant.it/

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!