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Plant Cell, 11ssue and Organ Culture 47: 293-297, 1997. ~) 1997 Kluwer Academic Publishers.

Printed in the Netherlands.


Research note

Somatic embryogenesis in liquid cultures of a tetraploid Alstroemeria

Margaret J. Hutchinson, T. Senaratna, J.M. Tsujita & Praveen K. Saxena*
Department of Horticultural Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 Canada (*requests for offprints)
Accepted in revised form 4 December 1996

Key words: cell suspension, morphogenesis, plant regeneration

Abstract A simple and efficient procedure was developed for regeneration of a tetraploid cultivar of Alstroemeria (A. pelegrina x A. psittacina) via somatic embryogenesis in liquid cultures. Embryogenic callus induced from mature zygotic embryos, cultured on MS medium supplemented with 40 /zM NAA and 2 0 / z M kinetin, was used as inoculum for liquid cultures. Pre-culture of the callus on MS medium supplemented with 80/zM NAA for two days was essential for cell proliferation in the liquid medium. Embryogenic cell aggregates, obtained by sieving through a 750 #m nylon mesh, continued to proliferate in media containing 10 or 20 #M NAA and 10 or 20/zM kinetin. When transferred to a semi-solid half strength MS medium supplemented with casein hydrolysate, cell aggregates successfully differentiated into plantlets which later grew to maturity under greenhouse conditions.

Abbreviations: MS - Murashige and Skoog (1962) medium; NAA - c~-naphthaleneacetic acid

Research on in vitro culture of higher plant cells and tissues has been stimulated, in part, by the prospect of using this technique for mass propagation, physiological and biochemical investigations of growth and differentiation as well as genetic manipulation of crop plants. Alstroemeria (Peruvian / Inca lily), a monocotyledonous crop belonging to the family Alstroemeriaceae, has gained world-wide importance as a cutflower, bedding and pot plant (Molnar, 1975). However, despite the limitations of traditional methods of propagating Alstroemeria and a considerable advancement in the development of in vitro techniques of plant production, progress towards micropropagation of this species has been slow due to its recalcitrant nature. To date, there are only few reports of plant regeneration from tissue cultures ofAlstroemeria. Gonzalez-Benito and Alderson (1992) and Hutchinson et al. (1994) reported morphogenic callus production from mature zygotic embryos of diploid and tetraploid cultivars of Alstroemeria, respectively. In this communication, we describe a simple and efficient method for proliferation of cells in liquid medium and the subsequent recovery of somatic embryos and whole plants in a tetraploid Alstroemeria cultivar (A. pelegrina x A. psittacina). To our knowledge, development of embryogenic suspension cultures in Alstroemeria has not been reported. Seeds of Alstroemeria used as the source material were kindly donated by Meyer Co. of Irvine, California, U.S.A. The seeds were scarified for 1 min in concentrated sulphuric acid and then rinsed three times with sterile distilled water. Seeds were surface sterilized by immersion, first in 95% ethanol for 5 min and then in commercial bleach solution (1.5% sodium hypochlorite) for 20 min with continuous stirring. To further soften the seed coat, seeds were soaked in sterile water in a sealed Petri dish for 48 h at 25 C in the dark. Imbibed seeds were re-sterilized using 70% alcohol and 1.5% sodium hypochlorite, as described above, before excision of mature zygotic embryos. Embryogenic callus was induced by placing the embryos on 25 ml of MS medium supplemented with 40/zM NAA with 20 # M kinetin in pre-sterilized Petri dishes (100 x 15 mm, Fisher Scientific Co., Unionville, Ontario).

294 The basal medium consisted of MS salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al., 1968) and 3% (w/v) sucrose. The pH of the media was adjusted to 5.7 and the solidifying agent (0.3% Gelrite; Schweizerhall, Inc., N J) was added before autoclaving at 1.19 kg cm -2 for 20 min. Four embryos were cultured in each Petri dish and three replicates were prepared for each treatment. The cultures were placed in the dark in a growth chamber set at 25 C. Ten two-month-old ealli pieces, approximately 1 mm 3 in size, were placed on MS medium supplemented with 80/~M NAA for two days and then transferred to 25 ml MS liquid medium supplemented with 10 or 20/zM NAA and 10 or 20 #M kinetin in 125 ml Erlenmeyer flasks. All liquid cultures were agitated at 150 r.p.m, on a horizontal gyratory shaker (G10 New Brunswick Scientific Co. Inc., New Brunswick, NJ) and incubated at 25 C in 16-h photoperiod (2025 #moles m -2 s -1) provided by cool white fluorescent tubes (Philips Canada, Scarborough, Ontario). The cultures were subcultured every five days. For subculturing, the liquid cultures were collected in centrifuge tubes and centrifuged at 75 g for 5 min to pellet the cell aggregates. The pellet was resuspended in 25 ml fresh medium and the suspensions filtered through a 750 #m sieve, and centrifuged again. The packed cell volumes (PCV) from the different treatments were recorded and one ml PCV of the fine cell fraction was transferred to 25 ml fresh culture medium. For plant regeneration, subculturing of the calli was discontinued and ten 4-week-old cell clusters (1-2 mm in diameter) formed in the cell suspensions developed above were transferred to a Petri dish containing semisolid (0.15% Gelrite) MS half strength medium, supplemented with 250 mg 1-1 casein hydrolysate. After eight weeks on the regeneration medium, the cell clusters differentiated into plantlets which were placed in Magenta boxes (Magenta Corporation, Chicago, IL) each containing 40 ml of semi-solid regeneration medium (same as above but with 0.3% Gelrite) for further development. Rooted plantlets were washed with tap water and transferred to 10 cm pots and two months later to 15 cm pots containing Pro-mix plug and germinating mix (Premier Brands Inc., Stamford, Ontario). Plants were maintained in the greenhouse and fertilized twice a week with 20:20:20 N.P.K. liquid fertilizer (250-280 ppm final concentration of N, P and K). Analysis of variance was performed on data collected using the general linear model procedure of the Statistical Analysis System (SAS Institute Inc., 1995). Calli induced from mature zygotic embryos of Alstroemeria on MS medium supplemented with 40 ~M NAA and 20 #M kinetin were used as explants to establish liquid cell suspension cultures. These growth regulator levels were determined empirically for optimal callus induction. The potential of this callus for developing embryogenic cell suspensions was evaluated using different NAA and kinetin concentrations (10 or 20/~M NAA and 10 or 20 # M kinetin). The calli obtained with 40/zM NAA and 20 #M kinetin were compact and did not disperse in any of the liquid media tested. However, placing the compact callus first on MS medium supplemented with 80/zM NAA for two days and then in the liquid subculture media (10 or 20 #M NAA and I0 or 20 #M kinetin) improved the friability of the callus, allowing for the production of finer cell suspensions (Figure 1A). The cell suspensions contained single cells, cell aggregates measuring about 0.5-2.0 mm in diameter and a few organized, but heavily vitrified structures (Figure 1A) that failed to develop further when transferred to regeneration medium. Cell suspensions of calli obtained following a'eatment with 80 #M NAA and then cultured continuously in a liquid medium without plant growth regulators failed to proliferate although they formed aggregates less than 1 mm diameter which did not regenerate (data not shown). After four weeks, most of the cultures maintained in basal liquid medium became necrotic and died. Incorporation of plant growth regulators in the liquid media increased PCV and the number of cell aggregates with a diameter between 1 and 2 mm after four weeks of culture. Cell aggregates smaller or larger than 1-2 mm in diameter were unsuitable for plant production due to poor growth, browning, and inconsistent regeneration. The fine cell suspensions that regenerated were formed at 10 #M NAA and 10 #M kinetin concentrations (Table 1). Although a higher auxin and cytokinin level (20 #M NAA and 20 #M kinetin) produced high PCV, the yield of fine cell suspensions decreased significantly as more aggregates per flask were formed, and kinetin had a stronger influence on the number of aggregates formed than NAA (Table 1). Four-week-old cell clusters, transferred to a semisolid regeneration medium, became nodular and somatic embryos at different stages of development were visible after about 14 days (Figure 1B, arrows). Well formed green shoots developed from these embryos after eight weeks (Figure 1C). More plantlets were regenerated from cell clusters from cell suspensions maintained in liquid MS medium supplemented


Figure 1. Somatic embryogenesis and plant regeneration from zygotic embryo-derived cell suspension cultures of a tetrapioid Alstroemeria (A. pelegrina xA. psittacina). (A) Cell suspension cultures maintained in liquid MS media supplemented with 10/zM NAA and 10/LM kinetin
and transferred to liquid MSO; For photography, suspensions were poured into a Petri dish (x0.7). (B) Development of earlier stages of somatic embryos (arrows) on semi-solid regeneration medium (x 1.1). (C) Somatic embryo-derived shoots (arrows) formed after 8 weeks of culture on semi-solid (0.15% gelrite) basal media (x 0.46). (D) Planflets regenerated from cell clusters three months after initial callus formation (x0.46).

with 20/zM NAA and 20 #M kinetin (Table 1; Figure 1D). The plantlets were successfully transferred to the greenhouse. The plants recovered from the cell suspensions showed no phenotypic variations from plants recovered from seeds and after ten months, these regenerants formed normal flowers.

Two requirements have been found to be critical to regenerating plants from cultures of most monocot crops, the presence of 2,4-D in the callus induction medium and the use of young meristematic tissues such as immature zygotic embryos, inflorescences, shoottip, and basal regions of young leaves (Prioli and Sondahl, 1989; Bhaskaran and Smith, 1990; KrishnaRaj

296 Tab/e 1. Effect of NAA and kinetin on Packed Cell Volume(PCV), cell aggregation, and plantlet formation from cell suspension cultures of a tetraploid Alstroemeria. NAA (#M) Kinetin (/zM) Total PCV 4SD (ml) Number of aggregates/ flask 4- SD 0 1.3 -4-0.56

Number of No. of aggregates plantlets/ regenerating aggregate 44- SD SD

0 0 10 tO I0 20 20

0 10 0 10 20 10 20

2.53 4- 0.50 2.92 + 0.56 2.15 4- 0.38 4.63 4- 0.75 6.30 4- 1.10 5.54 4- 0.03 7.11 +0.51

0 6.7 4- 3.06 7.7 4- 2.52 6.0 4- 2.00 9.3+ 1.16

0 6.0 4- 2.00 9.0 4- 4.36 7.7 4- 3.06 10.74-4.51

4.3 4- 3.10 12.3 4- 4.04 7.0 4- 1.00 11.04-3.61

(-) = not tested. Liquid cultures were started by transferring 1 ml PCV to 25 ml culture medium and growth (total PCV) and average number of aggregates (4- Standard Deviation, SD) per flask recorded after four weeks. The aggregates measuring 1-2 mm were counted; those less than 1 mm and greater than 2 mm were non-regenerable. Cultures consisting of ten 1-2 mm aggregates were scored for plantlet regeneration after eight weeks of culture and Vasil, 1995). In our study, zygotic embryos were used to d e v e l o p e m b r y o g e n i c callus. However, the calli were very compact, and in the resulting suspensions, cells tended to aggregate, especially in the presence of the cytokinin, kinetin. Yet the exclusion of kinetin in cell suspension cultures resulted in no or very poor regeneration o f plantlets. Regeneration in many monocotyledonous species has been shown to occur from compact callus that can be maintained in long-term cultures (Morrish et al., 1987) and friability is either difficult to achieve or results in non-regenerable cultures. The observation that placing induced calli on media containing a high N A A concentration prior to the transfer to regular maintenance media produced more friable callus is o f significance as the friable callus could be used to obtain relatively fine cell suspensions. The isolation o f friable embryogenic calli, especially in cereals, m a y allow the establishment of long-term solid and liquid cultures (Prioli and Sondahl, 1989). One possible reason for the N A A stimulated friability o f the callus m a y be the strong tendency of auxins to suppress organized growth (Schiavone and Cooke, 1987; K o m a m i n e et al., 1992). The observed role o f N A A in the induction o f somatic embryogenesis in Alstroemeria is notable as 2,4-D is the favoured growth regulator for cell proliferation in most monocotyledonous crops. Attempts to use 2,4-D in the present cultures was unsuccessful resulting in excessive browning and death of the explants (Hutchinson et al., 1994). In conclusion, a simple procedure for whole plant regeneration from cell suspension cultures o f a tetraploid Alstroemeria cultivar has been developed. Introduction o f a liquid phase significantly improved the efficiency o f regeneration as c o m p a r e d to those with callus cultures. Under optimal conditions o f the protocol earlier described, the callus derived from one zygotic e m b r y o produced two to six plants (Hutchinson et al., 1994) whereas in the present study, about 100 plants were produced by proliferating similar amounts o f callus under liquid culture. This procedure is expected to facilitate the study o f somatic e m b r y o g e n e s i s in Alstroemeria. The propagation o f e m b r y o g e n i c cells in suspension m a y facilitate bioreactor-based mass production methods of artificial seed development. We thank Fred M e y e r for providing the Alstroemeria seeds used in the study. This research was supported in part by an operating grant from the Natural Sciences and Engineering Research Council o f Canada to P.K.S. and the Canadian International D e v e l o p m e n t A g e n c y Scholarship to M.J.H.


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