Plant Cell, Tissue and Organ Culture 43: 287-289, 1995. 287. (~) 1995 ... Direct somatic embryogenesis and plantlet regeneration from cotyledonary leaves of ...
Plant Cell, Tissueand Organ Culture 43: 287-289, 1995. (~) 1995KluwerAcademic Publishers. Printedin the Netherlands.
287
Research note
Direct somatic embryogenesis and plantlet regeneration from cotyledonary leaves of safflower A.K.A. Mandal, A.K. Chatterji* & S. D u t t a G u p t a Applied Botany Section, Agricultural and Food Engineering Department, I.I.T., Kharagpur-721 302, India (* request for reprints) Received6 December1994;acceptedin revisedform 31 July 1995
Keyword: Carthamus tinctorius
Abstract Somatic embryos were induced directly on adaxial surface of cotyledonary leaves within 8-10 days of culture on Murashige and Skoog medium containing 5.37 to 10.74 laM 1 - napthaleneacetic acid and 2.22 p.M benzyl adenine. Germinated embryos with shoot axes developed into complete plants after transfer onto half stength Murashige and Skoog medium containing 1.07 p.M 1 - napthaleneacetic acid. Histological studies suggested direct origin of somatic embryos with broad-base attachment. Abbreviations: BA - benzyl adenine; MS - Murashige and Skoog; NAA - 1-napthaleneacetic acid; RH - relative humidity Safflower (Carthamus tinctorius L.) is a minor oil seed crop of semi-arid regions. The oil is valued therapeutically for its high degree of poly-unsaturation, and for its industrial use. Conventional breeding has helped in developing some elite cultivars, while in vitro technology could serve as an alternate means for further genetic upgrading, its successful application depends largely on a reliable plant regeneration system. Though substantial progress has been made in this direction in oil seed crops like Brassica (Maheswaran & Williams, 1986), Arachis (Hazra et al., 1989) and Helianthus (Finer, 1987), attempts in safflower have so far been restricted either to capitula induction (Tejovathi & Anwar, 1984), haploid callus induction (Rajendra Prasad et al., 1990) or shoot bud development (George & Rao, 1982; Tejovathi & Anwar, 1887; Chatterji & Singh, 1993). There has been no report of somatic embryo induction in safflower. This paper reports the induction of somatic embryos directly on the cotyledonary leaf surface and their regeneration potential.
Seeds of safflower (Carthamus tinctorius L.), cv. Girna (obtained from M.P.A.U., Maharastra, India) were surface sterilized with 0.1% mercuric chloride for 8-10 min, rinsed thoroughly in sterile deionized water and allowed to germinate on MS (Murashige & Skoog, 1962) basal medium with 0.8% Bacto-agar (BDH, India). Ten-day-old cotyledonary leaves were cut transversely into 5-8 mm long pieces and used as explants. They were aseptically placed on MS basal medium containing 3% sucrose and 0.8% Bacto-agar with the abaxial surface on the medium. The medium was supplemented with various concentraions of NAA (0.54, 1.07, 5.37 and 10.74 ~M) either singly or in combination with BA (2.22, 4.44. 8.87 and 17.74 ~M) and adjusted to pH 5.6 before autoclaving at 121 °C for 15 min. The cultures were incubated at 25 4- 1 °C under cool-white light with a 16-h photoperiod (36 ~mol m -z s -1) and 55-60% RH. Each treatment comprised 50 explants and the experiment was repeated thrice. Percent somatic embryogenesis (no. of explants with somatic embryos/no, of explants cultured × 100) and
288 number of somatic embryos per responding explant was calculated after 15 days of culture incubation. For complete plantlet regeneration, germinated embryos with shoot axes were transferred to halfstrength MS medium (gelled with 0.6% Bacto-agar) with 0.54, 1.07, 2.69 d 5.37 P-M NAA and incubated in the same cultural conditions. A total of one hundred somatic embryos with shoot axes were transferred to evaluate the conversion frequency. For histological studies, 15-day-old cultures with embryos were fixed in formalin, acetic acid and ethyl alcohol (1:1:18, v/v) for 48 h, dehydrated through graded series of ethyl alcohol and tertiary butyl alcohol and embedded in paraffin (58~50 °C). Serial sections of 10 p-m thickness were cut with rotary microtome and stained with 1% aqueous crystal violet solution. Explant swelling was apparent within 2 days of culture. Somatic embryos could be detected after 8-10 days on the adaxial surface of cotyledonary leaves. Within 15 days of culture, different developmental stages of the somatic embryos were detected (Fig. 1). The direct development of somatic embryos with their broad-base attachment could also be documented from histological studies (Fig. 2). Among the various NAMBA combinations tested, somatic embryogenesis was evident with 5.37 and 10.74 p-M NAA plus 2.22 p-M BA. With 5.37 p-M NAA in combination with 2.22 p-M BA, the embryogenic response was 30% and the mean number of somatic embryos per responding explant was 11.4 ± 3.2. However, a maximum embryogenic response of 54% with mean number of somatic embryos 14.7 + 4.1 per responding explant was obtained with the increase of NAA concentration from 5.37 to 10.74 p-M with 2.22 p-M BA. In contrast, higher concentrations of BA (4.44 to 17.74 p-M) along with NAA (0.54 to 10.74 p-M) favoured callusing without any somatic embryogenesis. NAA alone (0.54 to 10.74 p.M) did not induce somatic embryogenesis and at these concentrations rooting with limited callusing was observed at the cut end of the explant. Contrary to our observation,the combined effect of NAA and BA are reported to have induced only shoot buds on cotyledonary explants (George & Rao, 1982; Tejovathi & Anwar, 1987). This discrepancy in response may be due to the genetic variability of different cultivars as well as different approach of wounding of explants (Denchev et al., 1991) used for experiments. Such genotypic effects on regeneration is well documented (Jain et al., 1988). Germination of somatic embryos was evident with the emergence of shoots under the same cultural conditions after 21 days (Fig.
Fig. 1. Heart-shaped somatic embryo (arrow) emerging directly on a cotyledonary leaf after 12 days of culture on 10.74/aM NAA and 2.22 ~M BA. Bar = 0.5 cm. Fig. 2. Longitudinal section of a globular and a heart-shaped somatic embryo with notch of the heart (arrow) showing the broad-base attachment after 15 days of culture (e = cotyledonary leaf epidermis). Bar = 20 /am. Fig. 3. Germinated somatic embryo with shoot axis (arrow). Bar = h0 ram. Fi'g. 4. Regenerated plantlet.
3). At this stage the number of germinated embryos with shoots were found to be 2.8 -4- 1.1 per responding explant. Complete regeneration of plantlets was achieved after transferring these germinated embryos with a shoot axis onto half-strength MS medium containing 1.07 p-M NAA (Fig. 4). Conversion of germinated embryos into plantlets was 70%. Such a twostep regeneration protocol was also noted in Trifolium repens (Maheswaran & Williams, 1984). Among oil seed crops, direct somatic embryogenesis, has been reported in Brassica (Maheswaran & Williams, 1986), sunflower (Finer, 1987), peanut (Hazra et al., 1989) and niger (Sarvesh et al., 1993). In most of these cases, zygotic embryos were used as explant source. In the present study cotyledonary leaves, which are readily available throughout the year, were taken as the explant source. For the first time,
289 this s t u d y d e m o n s t r a t e s the s o m a t i c e m b r y o i n d u c t i o n d i r e c t l y f r o m c o t y l e d o n a r y l e a v e s o f safflower w i t h i n a narrow range of NAA and BA.
Acknowledgements A u t h o r s are grateful to M.P.A.U., M a h a r a s t r a , India, for s u p p l y i n g the seed. T h e first a u t h o r is also grateful to IIT, K h a r a g p u r , India, for p r o v i d i n g financial assist a n c e a n d to Prof. D . N . D e for l a b o r a t o r y facilities.
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