Somatic embryogenesis in cultured immature kernels of Pistachio, Pistacia vera L

PlantCell Reports

Plant Cell Reports (1995) 15:192-195

9 Springer-Verlag1995

Somatic embryogenesis in cultured immature kernels of Pistachio, P i s t a c i a vera L. A. Onay, C.E. Jeffree, and M . M . Yeoman University of Edinburgh, Institute of Cell and Molecular Biology, Daniel Rutherford Building, King's Buildings, Edinburgh EH9 3JH, U.K. Received 3 January 1995/Revised version received 29 March 1995 - Communicated by M.R. Davey

Abstract: Embryogenic tissue was produced from kernels o f immature fruits o f Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 m g r I casein hydrolysate, 114 p,M 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 gM benzylaminopurine. After transfer o f the embryogenic masses into the same medium, but with 4.4 pM benzylaminopurine, somatic embryos appeared. Several stages o f embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets. Abbreviations: 2,4-D - 2,4-dichlorophenoxyacetic acid; BAP benzylaminopurine (N6-benzyladenine); embryogenic mass EMS; MS - Murashige and Skoog medium (Sigma M-0404); NAA - el- naphthalene acetic acid; PGR - plant growth regulator; TDZ - thidiazuron (1-phenyl-3-(1,2,3, thiadiazol-5-yl)urea); WP McCown's Woody plant medium (Sigma M6774); ABA - abscisic acid Introduction Pistachio, Pistacia vera L., is a member o f the family Anacardiaceae which includes eleven species o f the genus Pistacia, all native to the middle east (Zohary 1952). Pistachio is an increasingly important nut crop, widely cultivated in hot dry areas of the eastern Mediterranean and the US. In the trade, Pistachio nuts are classified according to size, pericarp splitting (dehiscence) rate and kernel colour (Ayfer 1990). In general, small nuts are greener in colour, but those varieties with large green kernels have better aroma and texture making them desirable and expensive. Because Pistachio is a natural outbreeder, and is difficult to propagate by cuttings, clonal propagation is achieved by grafting buds from elite clones onto heterozygous rootstocks. Since the species is dioecious, it is common to see male and female scions grafted onto one rootstock. Incompatibility between rootstock and scion, frequently necessitates inter-grafting. Micropropagation thus offers a possible solution to the problems of clonally propagating elite Pistachio varieties. Growers do not have ready access to well-defined superior Pistachio strains, and research programmes have only begun recently to establish Correspondence to: A. Onay

new orchards of clonal material derived from Pistachio trees with proven superior characteristics. Most published research on micropropagation of Pistacia species has been carried out using juvenile or adolescent seedlings and less frequently on mature plants as source materials, (see Hansman and Owens 1986, Barghchi and Alderson 1989). However, juvenile plants cannot have revealed their fruit characteristics. Possible ways forward are to regenerate clones via somatic embryogenesis induced in material explanted from mature and proven elite clones, or from the embryos o f controlled crosses between parents with the desired characteristics. However, somatic embryogenesis is not easily achieved even from explants of juvenile material (Barghchi and Alderson 1985, Ahmad et al. 1994). The protocol presented here for the induction of somatic embryos directly from immature fruits represents the first stage of a study of the pathways of somatic embryogenesis in cultures of P. vera L.

Materials and Methods Source of plant material Immature fruits were used as a source of primary explants. These fruits were harvested in mid-June and early August 1993, approximately 8 and 12 weeks respectively after pollination, at Ceylanpinari state production farm, UrIh province, SE Turkey. Surface sterilisation. Immature kernels, from which the outer pericarp and shells had been removed, were pre-sterilised by immersion in absolute ethanol for 2 rain followed by a rinse with sterile distilled water. These pre-sterilised kernels were then exposed to 10% (w/v) hydrogen peroxide solution for 10 rain followed by a 20% (v/v) sodium hypochlorite solution (10-14% available chlorine) for 20 rain. The testas were then removed and the kernels washed three times with sterile distilled water before being placed in contact with the culture medium. Initiation o f ElliS. The auxins NAA and 2,4-D and cytokinins BAP and TDZ were tested in agar-solidified medium (agar, Sigma A1296; 0.7% w/v) at concentrations of 5.4 l-tM to 21.6 l-tM for NAA, 4.5 btM to 18 laM for 2,4-D, 4.4 to 35 p,M for BAP, and 4.5 to 36 laM for TDZ. Combinations of 2,4-D at 4.5 to 18 gM with BAP at 4.4 btM, combinations of NAA at 5.4 to 22 btM with BAP at 4.4 !,tM and treatments with BAP and TDZ were also tested in both MS and WP liquid media. Usually, the media were supplemented with 88 mM sucrose and adjusted to pH 5.7 before autoclaving. An EMS was initiated by culture of immature t?uits for 30 days in liquid MS medium with 200 mg I1 casein hydrolysate, 114 gM 1-ascorbic acid, 8.9 ~tM BAP, and 88 mM

193 sucrose. This EMS was then subcultured for 4 weeks on an embryogenesis expression medium having the same composition, but supplemented with 4.4 gM BAP. Subcultured explants were maintained in continuous light at 25~ in 250 ml culture tubes sealed with aluminium foil. Each treatment was initiated with 10 or 11 seeds. Maintenance and proliferation of EMS. The mother EMS thus obtained were transferred to a tissue proliferation medium that consisted of solidified MS medium supplemented with 500 mg 1"1 casein hydrolysate and 284 I,tM 1-ascorbic acid, either with 4.4 gM BAP or without growth regulators,. ]'he effects o f the carbon sources sucrose, glucose, fructose, lactose, ribose, xylose, mannitol, sorbitol and glycol on maintenance and proliferation were tested at concentrations of 58, 117, 175, 234, 292 and 351 mM. The cultures were incubated in continuous light (20 gmol m 2 s1) at 25 ~ and subcultured every two weeks. Maturation on agar-solidified medium: Ten days after subculture on the proliferation medium, pieces of actively-growing EMS were transferred onto agar-solidified MS medium with combinations of BAP at 4.4, 8,9, 18, 36, or 71 gM, and ABA at 1.9 gM together with sucrose at 58, 117, 175, 234 I,tM, and incubated at 25 ~ The embryogenic potential of the EMS is defined as the number of somatic embryos produced in 4 weeks per 250 mg fresh weight of EMS. Germination of somatic embryos. Germination studies were carried out in full-strength MS medium without PGRs, supplemented with 117 mM sucrose, 500 mg 1-~ casein hydrolysate, 284 laM 1-ascorbic acid and 0.7% agar. Transfer of plantlets to soil. In vitro germinated embryos were washed overnight in running water before being potted up in a sterile 1:1 mixture of peat and perlite or peat and grit. Plantlets were covered with a pyrex beaker to maintain 90+5% relative humidity for 4-5 weeks before transfer into glasshouse conditions (25~ day; 20~ night; 18-h daylength). Statistical analysis. A randomised complete block design was used for the initiation experiments. The Chi square (Z2) test was used to test the association effects of the model. In the maturation experiment, each treatment used two blocks, 5 replicates (Petri dishes) per block and 5 explants (ca. 50 rag) per replicate. A General Linear Model was performed and the t-test adjusted for the different number of observations used in pairwise comparison of treatments. Results and Discussion

Callus induction on agar medium. All o f the PGRs and combinations tested allowed some callus formation. Rapidly growing callus was initiated from immature kernels cultured on all media containing 2,4-D and NAA, but less callus was produced on media containing only BAP or TDZ. By the fourth week of culture, intact fruits gave rise to two distinct types o f callus, greyish yellow compact (Fig. 1A) or translucent creamy yellow nodular (Fig. 1B). Calluses initiated on agar-solidified media were maintained for more than one year by monthly subculture onto MS medium supplemented with 4.4 gM BAP, but expression of somatic embryogenesis was never observed from the callus initiated on agar-solidified medium. EMS induction in liquid medium. Initiation of EMS was influenced by fruit collection date and culture medium (Table 1). Statistical analysis showed no significant association between the BAP level and EMS produced on MS medium. The MS medium was found to be superior to the WP medium; kernels collected in early June did not show the same response. Among several PGRs and combinations tested, only three of the BAP treatments initiated friable embryogenic tissue, depending on the

concentration of BAP in the induction medium. The first stages of development of EMS visible on the external part of the explants were never observed before 4 weeks in culture. At the first subculture, EMS had formed in 20% of the 8.9 gM BAP treatments but in only 10% of the 4.4 gM and 18 gM treatments. The explants cultured on the highest concentration of BAP (36 gM) did not induce EMS and formed degenerate black tissue. The effectiveness of BAP in stimulating somatic embryogenesis from immature embryos has also been reported by Norgaard and Krogstrup (1991) who found that EMS was induced when explants of Abies nordmanniana were first grown on medium with a BAP content of 10 gM and subsequently maintained on a medium with the lower BAP concentration of 5 gM. In the present study EMS was induced from P. vera with a frequency of 20% with immature embryo explants cultured on BAP alone, but only in a liquid medium. In addition, the competence for somatic embryogenesis acquired in the presence of BAP was maintained after sub-culture on the same medium. Although the formation of EMS was greatest in MS medium with BAP, these EMS persisted on media containing sucrose with or without BAP. Proliferation of EMS. MS medium without PGRs proved to be the best proliferation medium for P. vera. However, if the culture was maintained for more than two weeks in the liquid medium, precocious maturation of somatic embryos was observed, resulting in the loss of the capacity to proliferate. EMS initiated in liquid medium containing sucrose could be maintained successfully in liquid medium with or without BAP. Therefore, subculture on the initiation medium without, or with a reduced level of the PGR used for initiation is a prerequisite for the maintenance of embryogenic tissues. Among the carbohydrates tested as sources of carbon, sucrose gave the best results. The best maintenance and proliferation was obtained in a medium containing 175 mM sucrose (data not shown). EMS transferred directly to a maintenance medium containing glucose, fructose, lactose, xylose, ribose, sorbitol, mannitol or glycol as alternative carbon sources lost some of their friability. EMS subeultured in liquid medium and the direct formation of somatic embryos. When embryogenic callus initiated on an agar medium was transferred to a liquid medium, all of the callus quickly browned and died. To enhance the growth and development of EMS and increase the frequency of globular embryos, EMS (Fig. 1C) formed on explants in liquid medium were selected and subcultured on a PGR-free medium or on media supplemented with BAP. The embryogenic tissues initiated and maintained in liquid media produced visible immature adventive somatic embryos within two weeks of the first subculture, which then developed further (Fig. 1D). These embryos were dark green at the globular stage. Subsequently, when the EMS were subcultured into PGR-free MS medium, or into MS medium containing 4.4 pM BAP, the original embryos continued to develop, and new embryos were also formed. When somatic embryos formed they were transferred directly onto MS agar or into liquid medium where about 90% of them doubled in size within 3 weeks. These swollen embryos were re-cultured on liquid MS medium either supplemented with BAP or without PGRs. Different patterns of development were observed in liquid medium

194

Fig.l. Somatic embryogenesis from immature seeds of Pistachio, Pistacia vera L.: (A) Greyish yellow compact callus after 4 weeks on solid medium (Bar = 3 ram). (B) Translucent creamy yellow callus after 4 weeks on solid medium (Bar = 3 mm). (C) Early stages of somatic embryogenesis after 4 weeks in liquid culture (Bar = 8 ram). (D) Developing somatic embryos after 6 weeks in liquid culture (Bhr = 2.5 ram). (E) Somatic embryos at various different stages of development 10 weeks after the culture was transferred to a liquid medium containing sucrose 87.6 raM, 1-ascorbic acid 114 gM and casein hydrolysate 200 mg 11, following separation by shaking (Bar = 2.5 ram). (F) Somatic embryo at the cotyledonary stage (Bar = 2 ram). (G) Germinated somatic embryos 2 weeks after the second subculture in liquid MS medium, showing the formation of globular adventive somatic embryos at the root tip (Bar = 4 mm). (H) A plantlet developed from a somatic embrye on an agar medium devoid of PGRs 3 weeks after transfer from liquid medium (Bar = 12 mm).

195 ten weeks after culture or two weeks after the second subculture (Fig. 1E). Many of the somatic embryos stopped growing at the cotyledonary stage (Fig. 1F) and some of them germinated with the formation of globular adventive somatic embryos at the root tip (Fig. 1G). Some other embryos developed into whole plantlets which matured in liquid medium (Fig, 1H). Table 1. Effect of seed collection date on initiation of embryogenic masses (EMS) from immaturefruits ofPistacia vera L. Collection date

Media

No. of immature fruits 1 Cultured With EMS

June 15th

MS 44 0 WP 44 0 August 15th MS 40 4 WP 44 0 1Data show only four of the cytokinin BAP treatments. Association between EMS and BAP level ~2 = 2.2 with 3 d.f. (not significant)) Maturation of somatic embryos on agar solidified medium

Morphological aspects. The colour of embryogenic tissue varied depending on the BAP concentration from yellowish green to deep green with some red pigmentation. Proliferative ability was influenced both by the sucrose and BAP concentration. EMS were proliferated in all treatments tested with sucrose and BAP, but showed reduction of growth rate, or partial inhibition of growth, on media with the highest concentration of BAP, 71 gM. By day 14 the effects of BAP and sucrose were evident not only in the inhibition of explants but also in morphological features. After 3 weeks somatic embryos appeared at stages of late embryogeny and started to develop cotyledons. By week 4, on media supplemented with 18 and 36 gM BAP, increasing the sucrose concentration from 58 to 234 mM resulted in precocious germination of somatic embryos. Especially in the medium supplemented with 18 gM BAP, half of the somatic embryos had germinated and had elongated radicles by week 4. At 4.4 gM BAP and 8.9 ktM BAP, precocious germination did not occur, but the white to yellowish somatic embryos showed elongation of the hypocotyls. With the concentration of BAP at 71 ~M the somatic embryos did not develop beyond the pre-cotyledonary stages unless they were transferred to fresh medium. However, with the concentration of BAP at 4.4, 8.9, 18 and 36 IxM somatic embryos developed well beyond the pre-cotyledonary stage when maintained in the same medium for a second 4-week period. Maturation of somatic embryos on agar-solidified medium Quantitative aspects. Of the treatments tested only sucrose and BAP had significant effects on the numbers of mature somatic embryos. Interactions between BAP concentration and sucrose concentration were evident (Table 2). Media with 18 gM BAP and 175 mM sucrose induced the most embryos (49 per 250 mg fresh weight). Increasing the BAP concentration from 18 to 71 t~M resulted in fewer mature somatic embryos. Sucrose concentrations above 175 mM were not beneficial. At the lowest BAP concentrations sucrose stimulated embryo production, but above 18 gM BAP, further increases in the sucrose concentration above 117 mM had an inhibitory effect, so that the highest concentrations of BAP and sucrose produced the lowest yields of somatic embryos. Germination of somatic embryos. Many of the somatic embryos developed abnormally with a reduced shoot but with well-grown roots. Most of the somatic embryos also produced new somatic embryos, and some embryos re-

callused depending on the germination medium. The effects of the BAP and sucrose concentrations on the germination of somatic embryos are currently being tested and will be reported elsewhere. Growth of plantlets. When somatic embryo-derived plants were successfully established in soil they showed normal growth after transfer to the greenhouse. However, some fungal contamination of the soil was observed, and the number of successful plantlet conversions from adventive embryos was low. Table 2. Numbers of mature somatic embryos per 250 rag fresh weight of Pistachio EMS produced on media with different BAP and sucrose concentrations after 4 weeks. Only visible mature somatic embryos with cotyledons were recorded. Means followed by the same lowercase letter are not significantly differentbased on Student's t-test Sucrose concentration(raM) BAP IxM 4.5 8.9 17.8 35.5 71.0

58 21.1 a 23.3 a 36.4b 14.9 c 13.1 c

117 26.8 a 39.8 b 49.2b 17.2 a 14.1 c

175 21.0 a 37.3 b 17.1 a 18.0 a 8.8 cd

234 35.4 b 43.8 b 16.4a 18.0 a 6.8 d

Conclusions. The initiation of EMS was observed only in the presence of BAP. Sucrose was the most favourable carbon source for maintenance of the embryogenic line. An important characteristic of the embryogenic line obtained was its capacity to proliferate embryogenic tissue at high rates (9fold at the 175 mM sucrose treatment in a relatively short time of 12 days) and, more importantly, to regenerate somatic embryos in the absence of growth substances (data not shown). The progress in Pistachio somatic embryogenesis described here shows promise as a model system for mass clonal propagation of Pistachio trees. Although somatic embryogenesis is a common feature of in vitro morphogenesis in many species of higher plants (see for example Mengol and Bagni 1992; Attree and Fowke, 1993), this appears to be the first report of the development of embryos to germination and beyond for P. vera L. The primary reason why this study has been based on seed explant material is that to date, little success has been obtained with the induction of embryogenic callus from mature explants. Consequently, efforts are now under way to initiate EMS from mature explant materials as well as to optimize the healthy germination of somatic embryos, and the acclimatisation ofplantlets. The establishment of a sound protocol for the production of somatic embryos of P. vera L. is the ftrst step towards a methodology for the production of clonal stocks. Acknowledgements. The researchwas funded by a grant fiom Universityof Dicle, Faculty of Science and Art, Diyarbaldr,Turkey, and performedunder ScottishOfficeAgriculturaland FisheriesDepartmentlicence.Gratefulthanksare extendedto Mr JohnFindlayof the ScienceFacultyElectronMicroscopeFacility forphotographyofthe tissuecultures. References

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