Agriculture Reference
In-Depth Information
genic mass to produce haploid plants. In brief, anther and ovule cultures are least
developed coffee tissue culture techniques despite their substantial potential in re-
ducing the time required to produce new varieties.
4.4 Citrus sp. (Family: Rutaceae)
Citrus
species represent the largest production of fruits worldwide. They are valued
for their antioxidant properties and are one of the richest sources of vitamin C and
essential oils.
The first report of development of haploid seedlings of
C. natsudaidai
was re-
ported by Karasawa (
1971
) by the application of gamma rays. Thereafter, there have
been several reports on production of haploids via in vivo crossing (Oiyama and
Kobayashi
1993
), gynogenesis induced by in vitro pollination with pollen from a
triploid plant (Germanà and Chiancone
2001
) and by in vivo parthenogenesis (Ger-
manà
2006
). Anther culture technique has been used to obtain haploid calli, embry-
oids and plantlets with limited efforts in a few
Citrus
species like
C. madurensis,
C. limon, C. deliciosa
x
C. paradise
(Chen et al.
1980
; Germanà et al.
1991
; Ger-
manà and Reforgiato
1997
). However, extensive research has been carried out on
C. clementina
. Since the first embryogenic calli and haploid plantlets were obtained
by anther culture in
C. clementina
Hort. Ex Tan. cv Nules (Germanà et al.
1994
),
many studies have been carried out to improve the androgenic response in
Citrus
species by the use of different combinations of plant growth regulators (Hidaka
et al.
1979
; Chaturvedi and Sharma
1985
; Geraci and Starrantino
1990
; Germanà
et al.
1994
,
2000a
,
b
,
2005
). Germanà and Chiancone (
2003
) proposed an improved
and detailed protocol for haploid induction through anther culture of
C. clementina
Hort. Ex Tan. cv Nules by evaluating a number of factors that affect androgen-
esis. They observed that temperature pre-treatment of flower buds between 4 °C
and 25 °C for 14 days was more favourable to induce embryogenic callus and em-
bryoids in anther cultures. Anthers were excised from pre-treated flower buds and
cultured on N
6
medium (Chu
1978
) supplemented with Nitsch and Nitsch vitamins
(Nitsch and Nitsch
1969
), galactose (9000 mg l
−1
), lactose (18,000 mg l
−1
), coconut
water (5 % v/v), casein hydrolysate (500 mg l
−1
), L-glutamine (200 mg l
−1
), biotin
(0.5 mg l
−1
), ascorbic acid (500 mg l
−1
), NAA (0.11 μM), 2,4-D (0.09 μM), Kinetin
(4.6 μM), BAP (2.2 μM), Zeatin (2.28 μM), TDZ (0.45 μM), and GA
3
(1.45 μM)
in dark for 15 days before being shifted to diffuse light at 25 ± 2 °C. With this pro-
tocol, 1.9 % anther cultures showed embryoid development and a total of 570 and
1,000 embryoids developed in Nules and SRA 63 cultivars, respectively. Direct
gametic embryogenesis without callus formation was observed in 7 % responsive
anther cultures of the cv Nules and in 45 % of the responsive anther cultures of the
cv SRA 63. The embryoids were later germinated on MS medium containing GA
3
(2.89 μM) and NAA (0.05 μM). Recently, Chiancone et al. (
2006
) studied the effect
of polyamines, spermidine and putrescine, with an aim to further improve the rate
of embryogenic callus and embryoid induction in anther cultures of
C. clementiana
