Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot

doi:10.1016/S0168-9452(00)00303-4

Plant Science

Volume 158, Issues 1–2, 8 September 2000, Pages 61-70

https://doi.org/10.1016/S0168-9452(00)00303-4 Get rights and content

Abstract

Relatively high percentages of adventitious shoot regeneration have consistently been obtained from leaves of some apricot cultivars. For the cultivar ‘Helena’, explants from the proliferation medium were more reactive than those from the elongation medium. The best results were obtained with thidiazuron (TDZ). When 6-benzylamino-purine (BAP) was used instead of TDZ, the regeneration percentages were very low. High α-naphthaleneacetic acid (NAA) concentration had an important effect upon the decrease of the secretion of phenolic substances. Young expanding leaves with the adaxial side touching the culture medium, maintained for 2 or 3 weeks in darkness, produced the best results. There was a significant genotypic variability in adventitious bud formation. Several caulinar meristems arose from very small areas of the leaf but only one meristem developed to form a shoot when those buds were transferred to elongation medium. However, the fact that several caulinar meristems exist in early steps could be an advantage when genetically transforming these leaves, since a high number of cells would have the possibility of being transformed and producing adventitious transformed buds.

Introduction

Adventitious regeneration is a key step in the application of genetic engineering techniques to the breeding of plants. Fruit trees are among the most recalcitrant to produce adventitious shoots, although reports on the successful regeneration and transformation of different species have appeared in recent years [1], [2], [3]. Reports of in vitro adventitious shoot regeneration from mature explants of Prunus are few. Adventitious shoots have been obtained from leaves of Prunus domestica [4], Prunus canescens [5], P. dulcis [6], P. persica [7] and P. serotina and P. avium [8]. In apricot (Prunus armeniaca L.), shoot regeneration through adventitious induction has been achieved frequently from juvenile explants or endosperm [9], [10], [11]. Because apricot is a highly heterozygous species, the use of seed-derived material should be avoided whenever elite clones have already been selected. Thus, it is important to establish effective protocols for induction of shoot regeneration from mature apricot explants. Only one paper about regeneration in apricot from adult tissue has been published to date [12]. The authors of that report found the results to be poorly reproducible.

The present study was designed to explore the conditions required for induction of adventitious buds and regeneration of shoots from explants of mature apricot trees propagated in vitro.

Section snippets

Maintenance of shoot cultures

This study has been carried out with the American cultivar ‘Helena’ obtained in the apricot-breeding program at the Horticultural Crops Research Laboratory in Fresno, California and kindly provided by Dr Craig A. Ledbetter. In vitro shoots were maintained by sub-culturing at 3-week intervals onto a shoot multiplication medium. This medium consisted of QL macronutrients [13] and micronutrients, vitamins and organic compounds as described previously [14], 3% sucrose and 0.6% agar (Hispanlab,

Results

The first change, observed within 7 days on regeneration medium, was the enlargement of explants to twice their original size. Morphogenesis occurred mostly on the cut edges and midribs or in association with vascular tissue. Most of the buds developed from calli, but sometimes appeared to arise directly from petiole tissue. The segments closer to the petiole were more regenerable than distal leaf segments. The first adventitious buds were observed 3 or 4 weeks after the beginning of the

Discussion

Many authors have observed that the majority of adventitious buds develop from calli in the basal cuts of leaves or from the petiole directly [12], [16], [17], which is in agreement with our results. Usually, the regeneration is associated with the cut side and the vascular tissue [18], [19], [20]. Sarwar and Skirvin [21] proposed that the cut edges provided a way for nutrients and growth regulators to be absorbed efficiently from the medium. Although most regeneration occurred from the calli

References (29)

V. Escalettes et al.
In vitro adventitious shoot regeneration from leaves of Prunus spp.
Plant Sci.
(1993)
E. Olmos et al.
Cellular adaptation from a salt-tolerant cell line of Pisum sativum
J. Plant Physiol.
(1996)
M. Welander et al.
Shoot regeneration from leaf explants of dwarfing apple rootstocks
J. Plant Physiol.
(1992)
D.J. James et al.
Factors affecting high frequency plant regeneration from apple leaf tissues cultured in vitro
J. Plant Physiol.
(1988)
M. Sarwar et al.
Effect of thidiazuron and 6-benzylaminopurine on adventitious shoot regeneration from leaves of three strains of ‘McIntosh’ apple (Malus x domestica Borkh) in vitro
Sci. Hort. Amsterdam
(1997)
M. Welander
Plant regeneration from leaf and stem segments of shoots raised in vitro from mature apple trees
J. Plant Physiol.
(1988)
I.V. Bartish et al.
The composition of nutrient medium and the efficiency of shoot induction in vitro from apple leaf explants
Russ. J. Plant Physiol.
(1997)
S. Sriskandarajah et al.
Conditioning promotes regeneration and transformation in apple leaf explants
Plant Cell Tissue Org. Cult.
(1998)
A.S. Trifonova et al.
Studies on genetic transformation of apple
Acta Hort.
(1999)
G. Bassi et al.
In vitro shoot regeneration on ‘Bluefre’ and ‘Susina di Dro’ prune cultivars (Prunus domestica L.)
Acta Hort.
(1991)

M. Antonelli et al.

The use of a brief 2,4-D treatment to induce leaf regeneration on Prunus canescens Bois

Acta Hort.

(1990)

C.M. Miguel et al.

Shoot regeneration from adventitious buds induced on juvenile and adult almond (Prunus dulcis Mill.) explants

In Vitro Cell. Dev. Biol. Plant

(1996)

V. Declerck et al.

Influence of growth regulators and carbon sources on callus induction, growth and morphogenesis from leaf tissues of peach (Prunus persica L Batsch)

J. Hort. Sci.

(1996)

N. Hammatt et al.

Shoot regeneration from leaves of Prunus serotina Ehrh. (black cherry) and P. avium L. (wild cherry)

Plant Cell Rep.

(1998)

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Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot

Abstract

Introduction

Section snippets

Maintenance of shoot cultures

Results

Discussion

Plant Sci.

J. Plant Physiol.

J. Plant Physiol.

J. Plant Physiol.

Sci. Hort. Amsterdam

J. Plant Physiol.

The composition of nutrient medium and the efficiency of shoot induction in vitro from apple leaf explants

Russ. J. Plant Physiol.

Conditioning promotes regeneration and transformation in apple leaf explants

Plant Cell Tissue Org. Cult.

Studies on genetic transformation of apple

Acta Hort.

In vitro shoot regeneration on ‘Bluefre’ and ‘Susina di Dro’ prune cultivars (Prunus domestica L.)

Acta Hort.

The use of a brief 2,4-D treatment to induce leaf regeneration on Prunus canescens Bois

Acta Hort.

Shoot regeneration from adventitious buds induced on juvenile and adult almond (Prunus dulcis Mill.) explants

In Vitro Cell. Dev. Biol. Plant

Influence of growth regulators and carbon sources on callus induction, growth and morphogenesis from leaf tissues of peach (Prunus persica L Batsch)

J. Hort. Sci.

Shoot regeneration from leaves of Prunus serotina Ehrh. (black cherry) and P. avium L. (wild cherry)

Plant Cell Rep.