In vitro shoot induction and multiplication from nodal segments of adult Ginkgo biloba plants

Abstract

The in vitro performance of herbaceous and woody nodal segments from adult plants and the effect of hydrolyzed casein (HC 500 mg L -1 ), kinetin (KIN; 6-furfurylaminopurine 0.46 and 4.65 µM) and activated charcoal (AC 1.5 g L -1 ) were evaluated upon new shoots induction and development, and to establish a system of in vitro propagation from adult plants of Ginkgo biloba. Woody nodal segments did not produce axillary shoots and presented 100% of bacterial and fungal contamination in culture. However, nodal segments from herbaceous shoots were successfully disinfected and displayed high in vitro morphogenic capacity. The HC was essential for the axillary shoots induction and further multiplication, stimulating shoot formation in 85% of the cultured nodal segments and multiple shoots induction in 35% of them at establishment stage. During the multiplication stage, 66.6% of propagules formed new shoots and 33.3% of them formed multiple shoots when cultured with HC. The KIN and AC inhibited the organogenic process in ginkgo. Two distinct patterns of sprouts development were observed in vitro, similar to what occurs in vivo: 1) short shoots with crowded internodes and expansion of only a few leaves and slow growth; 2) long shoots with separated nodes and marked apical growth. This is the first report of multiple shoots in vitro formation in nodal segments obtained from adult plants of Ginkgo biloba.

The efficient method for in vitro micropropagation of Ginkgo biloba L.

“…In the literature, in vitro regeneration of G. biloba is mostly limited to cultures of intact embryos [18][19]. There are also some reports regarding applying male gametophytes [20], female gametophytes [21], nodal segments and buds [9,[11][12] as explants for in vitro culture and micropropagation of this medicinal plant. At the current study, among stem, leave and lateral bud as explants, only the last one showed positive response to in vitro regeneration.…”

“…In this study, the treatment containing Kin (1 mg/L) + IAA (0.5 mg/L) has shown the best response regarding shoot length (2.47 cm) and leaf number per explant (6.5). While Tommasi and Scaramuzzi [11] reported callus induction and low shoot regeneration by applying IAA + Kin (in MS basal medium with endosperm extract) and Mantovani et al, [12 ] showed that hydrolyzed casein is essential for the axillary shoots and future mollification. The assessment of the effects of different cytokinins on shoot multiplication of ginkgo showed that Kin promoted the formation of multiple shoots [12].…”

“…While Tommasi and Scaramuzzi [11] reported callus induction and low shoot regeneration by applying IAA + Kin (in MS basal medium with endosperm extract) and Mantovani et al, [12 ] showed that hydrolyzed casein is essential for the axillary shoots and future mollification. The assessment of the effects of different cytokinins on shoot multiplication of ginkgo showed that Kin promoted the formation of multiple shoots [12]. Results of other researchers have also shown that the Kin increases shoot number proliferation.…”

“…However, nodal segments from herbaceous shoots were displayed high in vitro morphogenic capacity. Results also demonstrated the essential role of hydrolyzed casein for the axillary shoots induction and further multiplication [12]. In another study, different developmental stages of immature embryos of G. biloba were cultured on MS medium supplemented with various 2,4-D, NAA, BAP and zeatin or with various concentrations of 2,4-D alone.…”

“…There are some difficulties in generative propagation of G. biloba including dioceous nature, long juvenile phase, long reproductive cycle, and low germination rate, short period of seeds viability and recalcitrant nature of seeds [11]. Otherwise, the vegetative propagation through cuttings has low income and is extremely dependent on the juvenile nature of explants [12]. In order to overcome referred obstacles and to conduct a massive and rapid production scheme of this medicinal plant, in vitro propagation were used as an alternative and effective way in previous studies.…”