Plant Science 159 2000 213 – 222
In vitro selection and characterisation of a drought tolerant clone of Tagetes minuta
M.A.-H. Mohamed
a,b
, P.J.C. Harris
a
, J. Henderson
a,
a
School of Natural and En6ironmental Sciences, Co6entry Uni6ersity, Priory Street, Co6entry, CV
1 5
FB, UK
b
Department of Horticulture, Faculty of Agriculture, Mania Uni6ersity, El-Mania
61111
, Egypt Received 5 July 1999; received in revised form 22 May 2000; accepted 7 July 2000
Abstract
Aromatic Tagetes plants produce secondary products which have a biological activity against a wide range of micro-organisms, insects and nematodes. Tagetes oils are also used as pharmaceuticals and as flavour components in the food industry. This study
aimed to use somaclonal variation to select drought tolerant plants of Tagetes. Cotyledons cultured on MS medium containing 3 mg l
− 1
IAA and 10 mg l
− 1
BA callus growth medium; CGM with 60 mM mannitol died. Shoot clumps developed on CGM for 6 months and then subcultured onto CGM containing 80 mM mannitol also died. Four shoots were regenerated from 72 shoot
clumps on
1 2
MS medium containing 0.5 mg l
− 1
IAA shoot growth medium; SGM after culturing on CGM without mannitol for 6 months and then on CGM with 60 mM mannitol for 3 months. Twelve shoots developed from 72 shoot clumps on SGM
after culture for 9 months on CGM. Significant variations were observed in biomass amongst regenerated clones when cultured on medium containing mannitol. After growth in greenhouse conditions for 2 months, one clone developed from shoot clumps
selected on medium with mannitol exhibited a significant tolerance in vitro in medium containing 90 mM mannitol; this medium completely inhibited growth of control plants. This clone had significantly higher proline content and soluble sugars than the
non-stress-selected clone when cultured on medium containing 0 or 30 mM mannitol. When tested for drought tolerance growth at 40 soil field capacity in the greenhouse for 2 months, this clone showed a significant tolerance compared with other
regenerated and control plants and revealed lower water potential, greater accumulated biomass and a higher relative growth rate. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Marigold; Water stress; Somaclonal variation; Regeneration www.elsevier.comlocateplantsci
1. Introduction
The importance of Tagetes minuta L. Mexican marigold Asteracea as a source of volatile oils
and thiophenes as well as its practical uses have been reviewed [1 – 3]. Volatile oils of T. minuta
have a biological activity against a wide range of microorganisms and insects. They are also used in
perfumes and as flavour components in many food products. The plants have a suppressive effect on
free-living nematodes and have been used as an intercrop or in rotation to protect crops [4].
Water deficit is a major component of environ- mental stresses such as drought, salinity and low
temperature, and 40 – 60 of the agricultural land around the world suffers from drought [5,6].
Breeding for water stress tolerance by traditional methods is a time consuming and inefficient proce-
dure [7]. In vitro culture may be used to obtain drought-tolerant plants assuming that there is a
correlation between cellular and in vivo plant re- sponses [8]. This method is based on the induction
of genetic variation among cells, tissues andor organs in cultured and regenerated plants. Al-
though there are genetic, biochemical and physio- logical constraints in obtaining stress-tolerant
Abbre6iations
:
BA, 6-benzyladenine; CGM, callus growth medium; FC, field capacity; IAA, indole-3-acetic acid; RGR, relative growth
rate; SGM, shoot growth medium. Corresponding author. Tel.: + 44-24-76888632; fax: + 44-24-
76888702. E-mail address
:
janey.hcoventry.ac.uk J. Henderson. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 3 9 - 3
plants through in vitro culture, Nobars [6] pointed out that this technique has been successfully used
to produce stress-tolerant plants from several species.
The accumulation of proline and soluble sugars as an osmotic tolerance mechanism has been
widely observed in many species. Proline may provide
osmoregulation and
stabilisation of
proteins and membrane during stress [9]. Soluble sugars, which accumulate in the vacuole, are a
major organic solute, involved in osmotic adjust- ment when plants are exposed to drought [9].
Sucrose, mannitol or sorbitol were studied by Sabili et al. [10] as osmotic stress agents on in
vitro-grown Chrysanthemum morifolium. Sucrose failed to elicit consistent osmotic stress symptoms
and enhanced both shoots and root growth. The osmotic potential of the tissue paralleled the in-
crease of mannitol or sorbitol concentrations on culture medium. The harmful effect of the osmotic
stress agent occurred in both shoot proliferation and rooting stages. Sorghum bicolor was tested in
vitro for drought stress by Dunca et al. [11]. Results showed that osmotic stress applied to in
vitro cultures reduced regeneration ability. How- ever, by screening the regenerated plants under
field conditions they obtained stress-tolerant re- generates with higher yield under stress conditions
than that of their parents.
Regeneration of plants displaying an increased tolerance to environmental stress is an important
goal for the biotechnological improvement of many plant species [6]. Therefore, the aim of this
investigation was to induce somaclonal variation in regenerated plants in order to select drought-
tolerant clones of T. minuta, which grows as a cash crop in some developing countries [2,3], using
a rapid in vitro regeneration protocol [12].
2. Materials and methods
