EFFECT OF GENOTYPE ON SOMATIC EMBRYOGENESIS IN PISTACHIO (PISTACIA VERA L.)

Propagation of Omamental Plants Vol. 7, N° 4, 2007: 204-209

EFFECT OF GENOTYPE ON SOMATIC EMBRYOGENESIS IN PISTACHIO (PISTACIA VERA L.) Ahmet Onay1, Engin Tilkat1*, and Hakan Yildirim2 'Dicle University, Faculty of Science and Letters, Department of Biology, 21280-Diyarbakır, Turkey, *Fax: + 90 412 248 80 39, *E-mail: [email protected] 2 Dicle University, Faculty of Agriculture, Department of Horticulture, 21280-Diyarbakır, Turkey Abstract Genotypes representing eight botanical cultivars of pistachio (Pistacia vera L.) were assessed for somatic embryogenesis and subsequent plant conversion from immature kernels. Embryogenic masses (EMSs) were produced from kernels of immature fruit of pistachio cultured in liquid MS medium with Gamborg vitamins, supplemented with 100 mgl1 casein hydrolysate, 100 mg l-1 L-Ascorbic acid, 3% sucrose, and BAP. EMS dif-ferentiated directly from the kernels after culture for 2 weeks in liquid medium with 0.5 mg l-1 BAP. Genotype x collection date had large effects on initiation of somatic embryogenesis. Embryogenic tissues containing em-bryoid or individual somatic embryos were isolated from the original tissue and used to initiate repetitive liquid or agarified cultures. To determine the effects of genotypes during maturation on the subsequent germination and plantlet generation, clusters of EMSs from the different genotypes were transferred from liquid medium onto the surface of 5.7% ağar solidified MS medium with Gamborg vitamins, containing 4% sucrose, 1.0 mg l1 BAP and 0.5 mg l-1 IBA. After 4 weeks, individual or clusters of mature somatic embryos were transferred from maturation medium onto the germination medium (GM). The number of matured and germinated somatic embryos was influenced by the genotype in the maturation medium. Acclimatized plantlets resumed growth after transfer to a soil and peat mixture, and many developed to maturity. Key words: genotype effect, pistachio, plant regeneration, somatic embryogenesis

INTRODUCTION Pistachio, Pistacia vera L. is a member of the family Anacardiaceae, which includes eleven species of the genus, ali native to the Middle East (Zohary 1952). 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. Therefore, the expansion of pistachio plantations by the use of traditional methods has been limited. One solution may be to regenerate clones via somatic embryogenesis induced in material explanted from selected mature fruit-bearing pistachio trees old enough to have expressed their superior charachteristics. in pistachio we have standardized the efficient protocols for direct somatic embryogenesis from immature fruits (Onay et al. 1995, 1996, Onay and Jeffree 2000) and mature zygotic embryos (Onay 2000). The protocol involves multiple stages, including embryogenic mass formation, maturation, germination, and plantlet development. One of the important factors that affect embryogenic response in the somatic tissues of plants is the genotype. With this factor in mind, a

Received: October 1, 2007

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great deal of research has been conducted and reviewed in several important plant species (Steger et al. 2003, Mackay et al. 2006). However, none of the existing reports in pistachio describe the genotype-specific response at each stage of somatic embryogenesis. in the investigation presented here, the genotypic control of somatic embryogenesis in pistachio using a single protocol initially optimized for pistachio (Onay et al. 1995) has been examined with 8 additional genotypes. MATERIALS AND METHODS Explant source immature fruits of eight genotypes of pistachio were used as a source of primary explants. These fruits were harvested in June 15, July 1 and July 15, 2006, approximately 8, 10 and 12 weeks, respectively after pollination. Surface sterilization immature kernels (Fig. 1.1), from which the outer pericarp and shells had been removed, were exposed to a 10% (v/v) sodium hypochlorite solution (Com-

Accepted: November 10, 2007

Ahmet Onay et al. Effect ofgenotype on somatic embryogenesis in Pistacia vera L.

Germination of somatic embryos

mercial Auxion) for 5 min. The testas were removed and then the kernels washed thrice with sterile distilled water before being placed in contact with the culture medium.

Media used for germination are presented in the Table 1. Eight to twelve embryos with elongated hypoco-tyls, restricted plumules and well-developed roots were transferred to the media that had been optimized for plant development. Ali the produced somatic embryos, irrespective of shapes and sizes were used randomly for these experiments. The experiment was repeated twice. Germinated somatic embryos developed into plantlets on the same medium. A plantlet was counted when the hypocotyls elongated. Ali the cultures were incubated at 25° ± 2°C under white-florescent light at photosynthetic photon flux of 35-40 jımol m-2 s-1 and a 16-h photoperiod. in ali media, the pH was adjusted to 5.7 prior to the addition of ağar, and the media were autoclaved at 121 °C for 15 min.

Initiation of embryogenic masses The methods described by Onay et al. (1995) for somatic embryogenesis in immature fruits of pistachio were followed for the induction of embryogenic celi masses, development of somatic embryos, radicle emergence/embryo conversion and plant development. Fifty kernels from each of the 8 genotypes were cultured for 3 weeks in liquid embryogenesis induction medium (EIM). The composition of media used in the experi-ments is given in Table 1. After 2 weeks, the number of explants that had produced EMS in each Erlenmeyer fiask was recorded. Maintenance and proliferation of EMS The mother EMS thus obtained were transferred to MS (Murashige and Skoog 1962) medium having the same composition (EIM) but supplemented with 0.5 mg l1 6-benzylaminopurine (BAP) for 2 weeks. Subcultured explants were maintained in 250 mi culture tubes, containing 50 mi of EIM medium (5 explants (ca. 250 mg per flasks)), sealed with aluminium foil.

Acclimatization and 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 soil. Plantlets were covered with a Pyrex beaker to maintain 90 ± 5% relative humid-ity for 4-5 weeks before transferred into glasshouse conditions (20°C, 16-h day length). They were kept under greenhouse conditions four weeks before being transferred to outdoor conditions.

Maturation on agar-solidified MS medium Statistical analysis The cultures in ali experiments were set out in a completely randomized block design. For initiation experiments, each treatment consisted of 50 immature fruits, with the experiment repeated twice. Four to six explants (immature fruits) were added to each Erlenmeyer fiask. After 3 weeks of incubation in liq-uid medium, immature fruit producing embryogenic masses was recorded. The number of mature SEs per one gram EMS was recorded after 4 weeks of culture. The number of plantlets that developed from rooted embryos in media was assessed after 4 weeks of incu-

Two weeks after subculturing on proliferation medium, pieces (5 replicates (Petri dish) per block, 4 explants (ca 250 mg per replication)) of actively growing EMS were transferred onto embryo maturation medium (EMM, Table 1). The experiment was repeated twice. The embryogenic potential of the EMS is defined as the number of somatic embryos produced in 4 weeks per 1 g fresh weight of EMS for each of studied genotype. Somatic embryos (SE) > 5 mm long, with visible shoot and root axes were considered as mature.

Table 1. Composition of media used in the experiments. Constituent

Basal medium

Embryogenesis Đnduction Medium (EIM)

Embryo Maturation Medium (EMM)

Germination Medium (GM)

MS macro- and microelements

+

+

+

+

+

+

+

+

L-Ascorbic acid (mg M)

100

100

100

100

Casein hydrolysate (mg M)

100

100

100

100

Sucrose (g M)

30

30

40

40

-

-

5.7

5.8

0,5

0.5

-

0.5

1 2

Gamborg's vitamins

Ağar (g M) BAP(mg M) ABA (mg M) 1

-

2

Murashige and Skoog (1962), Gamborg et al. (1968)

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Propagation of Omamental Plants Vol. 7, W° 4, 2007: 204-209 bation. Data presented in Tables 2-3 are mean values of the replicated experiments. For EMS initiation, a general linear model (GLM) was constructed and least signifıcant difference, adjusted for the different number of observations was used for multiple comparisons. For somatic embryo maturation and plantlet conversion Kruskal Wallis Test was applied and Tukey's HSD was used for multiple comparison. The term "conversion" implies the spontaneous differentiation of somatic embryos to plantlets with shoots and roots, whereas the induced differentiation of plumule is hereafter termed as plant development. RESULTS AND DISCUSSION EMS induction in liquid MS medium initiation of EMS was influenced by fruit collection date and genotype (Table 2). There were signifıcant dif-ferences in the frequencies of EMS obtained between the genotypes tested (Table 2, p < 0.01). in ali of the genotypes tested, EMS did not appear vvithin 2 weeks of the culture of immature fruits being initiated. During the first week on liquid medium the outer epidermal cells of most of the cultured intact immature fruits became ablack-brown circle. After 2 weeks culture, the major-ity of the explants increased in size and were cultured onto a fresh EIM. immature fruits collected on July first show similar response rates with the explants collected in June 15th and the response rate was lowered with the explants harvested in July 15th, but June 15th was found to be superior to the other collection dates tested. Nodular structures were developed within the EMS of the fıve genotypes ('Uzun', 'Kırmızı', 'Siirt', 'Ker-man', and 'Ohadi') which, after three subcultures on MS medium containing 0.5 mg l"1 BAP, appeared to be entirely composed of embryogenic calluses (Fig. 1.2). The frequency of EMS induction varied from 7% for

genotype 'Kerman' to 19% for 'Uzun' (Table 2). The effect of genotypes on embryogenic tissue induction has also been demonstrated in some conifer species (Jain et al. 1989, Cheliak and Klimaszewska 1991). Proliferation and maintenance of EMS and the direct formation of somatic embryos Önce an EMS initiated in liquid medium containing 3% sucrose and with or without (after a few subcultures) 0.5 mg l1 BAP it could be maintained successfully in liquid or the agarifıed medium. Therefore, subculture on the initiation medium with BAP used for initiation is a prerequisite for the maintenance of embryogenic tissues (Onay et al. 1995). If the EMS was maintained for more than two weeks in the liquid medium and transferred to EMM, most of the somatic embryos swol-len (Fig. 1.3). Many of the somatic embryos stopped growing at the cotyledonary stage and some of them germinated with the formation of globular adventive somatic embryos at the root tip. Some other embryos developed into whole plantlets, which matured in liquid medium. High percentage of abnormalities in pistachio somatic embryos was frequently observed in previous studies (Onay et al. 1995). The ability to form somatic embryos is considered to be a trait under genetic control, and the individual genotypes within a species can dif-fer in their ability to undergo somatic embryogenesis (Parrott et al. 1991). Maturation of somatic embryos on ağar solidified medium Genotype had a signifıcant effect on the number of mature somatic embryos observed (Table 3). 'Kırmızı' and 'Siirt' induced the most somatic embryos (153 and 148 per 1 g fresh weight, respectively). 'Uzun',

Table 2. Effect of fruit collection date on initiation of embryogenic masses (EMS from immature fruits of P. vera L.). The data were recorded after 14 days in culture. % of fruits with embryogenic mass (EMS) Genotypes collection date

June 15th

July first

July 15th

'Kırmızı' 'Siirt' 'Uzun' 'Halebi' 'Sultani' 'Vahidi' 'Ohadi' 'Kerman' Source Model Genotype Duration

8 11 19 0 0 0 8 7 F 15.781 18.243 5.936

9 9 12 0 0 0 6 5 P-value 0.000 0.000 0.014

4 5 4 0 0 0 0 5

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Ahmet Onay et al. Effect of genotype on somatic embryogenesis in Pistacia vera L.

Table 3. Effect of different genotypes on the numbers of matured, germinated SEs and plantlet regeneration per lg fresh weight of pistachio EMS. Genotype 'Kırmızı' 'Siirt' 'Uzun' 'Kerman' 'Ohadi'

No. of mature 12 SEs 153 ±2.76 a 148 ±2.53 a 70 ± 2.54 c 78 ± 1.25 c 96 ± 2.08 b

2

No. of germinated SEs(%)

Plantlet regeneration (%)

100 100 100 100 100

21 ± 1.33 b 14 ± 0.70 c 25 ± 0.98 a 14 ± 0.76 c 10 ±0.70 d

1

Matured SEs with cotyledons were counted by visual observation after 4 vveeks of culture. Means followed by the same letter are not significantly different at p < 0.05 probability level based on Tukey's HSD test.

2

'Kerman', and 'Ohadi', produced significantly less somatic embryos (70, 78 and 98 per 1 g fresh weight, respectively). Genotype-dependent embryogenesis was reported in Arachis hypogaea L. (Ozias-Akins et al. 1992) and Cajanus cajan L. (Venu et al. 1999). EMSs produced in liquid medium were transferred to embryo maturation medium. By day 7, pieces of actively grow-ing EMS transferred to the EMM had increased in size enormously. By day 14, the effects of genotype were evident not only in the inhibition of explants but also in morphological features. After 2 weeks of culture, the developing somatic embryos started developing elongated cotyledons. None of the treatments tested produced any precociously germinated SEs during 3 weeks of culture in the agarified EMM. After 3 weeks somatic embryos appeared at stages of late embryogeny and started to develop cotyledons. Nearly half of the somatic embryos had germinated and had elongated radicles by week 4. Precocious germination of somatic embryos, especially in 'Kırmızı' and 'Ohadi' genotypes was very often evident. Precocious germination of'Siirt' and 'Uzun'genotypes did not occur, but the white to yellowish somatic embryos showed elongation of the hypocotyls. Ali the somatic embryos of ali genotypes developed well beyond the pre-cotyledonary stage when maintained in the same medium for a second 4-week period. Mature somatic embryos were bipolar with a radicle and cotyledons (Figüre 1.4). Germination of somatic embryos Radicle differentiation of the pistachio somatic embryos raised from immature fruits is genotype-inde-pendent, whereas plumule differentiation is infiuenced by genotype. Embryos of ali 5 genotypes had radicle emergence on plant growth regülatör (PGR)-free basal medium (Table 3). Ali of the isolated mature somatic embryos produced plantlets on GM (Figüre 1.5A) but when clusters of mature somatic embryos were transferred to GM (Figüre 1.5B), some 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. Some embryos became swollen, and these embryos produced second-

ary embryos when they were re-cultured on EIM. Such abnormalities have been frequently observed by Onay et al. (1995,2007). Plantlet regeneration in ali genotypes, shoots and roots differentiated si-multaneously in PGR-free medium to produce plantlets by the usual process of meristem differentiation, as in zygotic embryos (Table 3, percentage of conversion). However, the frequency of conversion was relatively low and varied from 10% in 'Ohadi' to 25% in 'Uzun', in ali genotypes root differentiation was achieved in germination medium. Cultivar 'Uzun', which showed the highest frequency of embryogenic mass formation (19%>), exhibited the highest conversion frequency. in contrast, 'Kerman', which ranked fifth for embryogenic mass formation (7%>), had a conversion frequency of 14% upon transfer to PGR-free basal medium. Evidence suggests that similar to the embryogenic response, conversion ability is also under genetic control. How-ever, failure in pistachio somatic embryos to undergo conversion to plants is primarily due to morphological abnormalities (Onay et al. 1995), including incomplete maturation (Onay et al. 1997, 2000). Of the somatic embryos formed from mature zygotic embryos of pistachio cultivar 'Uzun', 70% matured in the same medium and converted into plantlets (Onay et al. 2007) in medium devoid of growth regulators. in contrast, when the embryos of this genotype were generated from immature fruits, only 25% converted (Table 3). These observations indicate that the conversion frequency of somatic embryos depends not only on the origin of the explants but also on various other mediating fac-tors. it is not clear whether these factors are controlled directly or indirectly by the genotypic constitution of the plant. Irrespective of the genotype, the frequency of plant development could be improved by extending the incubation period to 6 weeks (data not shown). However, the relative rank of genotypes after 6 weeks of plant development was not ahvays parallel to the ranks obtained in 4 weeks. An optimum response in each genotype could be achieved by specific refinement of the protocol. Onay et al. (2000) reported that the shoot

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Propagation of Ornamental Plants Vol. 7, Jfa 4, 2007: 204-209

Fig. 1. Somatic embryogenesis in cultured immature fruits of Turkish commercial cultivars. 1.1) immature kernels (explanted in June 15th) of pistachio, P. ver a L. Bar = 5 mm, 1.2) EMS initiated from immature fruits on EIM supplemented with 1 mg l"1 BAP after 3 subcultures and developed on embryogenic EIM containing 0.5 mg l1 BAP, Bar = 25 mm, 1.3) Swollen somatic embryos after 4 weeks on the agarified MS medium in the presence of 0.5 mg l1 BAP, if the EMS were maintained more than two weeks in liquid medium, Bar =10 mm, 1.4) Mature somatic embryos developed from the EMS after 5 weeks EMM containing 0.5 mg l1 BAP and 0.5 mg l1 ABA, Bar = 21 mm, 1.5) Germination of isolated (A) and clusters of (B) somatic embryos 28 days after transfer to germination GM devoid of PGRs, showing the formation of globular adventive somatic embryos at the apical tips (see arrows), Bar =12 mm, 1.6) Somatic plantlets raised by conversion of the germinated somatic embryos 10 months after transfer to soil, Bar =100 mm. differentiation was increased further by extending the incubation period from 4 to 6 weeks. in vitro regener-ated plantlets, after four or five weeks, hardening in peat and soil (Fig. 1.6) were transferred to greenhouse conditions. Acclimatized plantlets resumed growth after

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transfer to a soil and peat mixture, and many developed to maturity. immature fruit explants of pistachio initiate EMS readily. Further exploitation of this system for mass propagation and for regenerating transgenic plants could assist in the clonal propagation of pistachio.

Ahmet Onay et al. Effect ofgenotype on somatic embryogenesis in Pistacia vera L.

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