Techniques for in vitro seed germination in Pistacia species

A reliable technique for achieving in vitro seed germination in Pistacia vera cv. mateur, P. atlantica, P lerebinthus and P. /entiscus L. is documented . Both eradication of browning exudate from seeds and germination percentage were affected by the scarification of the seeds with hot water and sulphuric acid. Moreover, in-vitro germination of seeds and e longation of the seedlings were op timal when the seeds were cultured on modified Heller medium (H + 7 mM KH 2P04 + 1 0 ~M AgNO, ).


Introduction
Pis/acia species belong to the AnacQrdillCeae fam il y and are considered as one of the m ore important plants in many regions: Magreb, Spain, Turkey, Greece, Iran, Iraq, India. The propagation of these spec ies by seeds is quite difficult as the seeds are sensitive to desiccation and can not be stored for long periods w ithout a se rio us loss o fvi"b ility. Ti ssue culture techniques may be an alternative methodology for propagati on of Pislaeja _species. Howeve r, hig h co ntam ination rates are o ften observed ill plant tis sue cultures initiated from exp lants of Pistacia species taken from natural habi tats. Exp lants tak en from Pistaeia vera cv. malellr, P. allcmOca and P. terebilllhus seed lings and collected from greenho use were found to be highl y contaminated w ith Fusarium species (Mederos el 01.,unpubli shed data). For this reason in this study we investigated a method for reducing contami natio n fro m th ese Pistacia species. In an earlier study on in vdro shoot form ation from seedlings of these species we o bserved browni ng exudate to the cul ture med ium. Ou r results ind icated that the brown ing o f Pis/acia species seeds is related to the inhibition of in vitro germi nation (Mederos 199 1) and for th is reason eradication of oxidation of the pheno li c exudate was attempted. This study is a part o f a project aimed at fi nding a meth od to propagate Pis/acia s pecies ill vitro using seedling mater ial.  (1993).

Scarification treatment
Seeds from these speci es were placed separately in a gauz!! sack and su bmerged completely in hot water for 15 and 25 min at 33°C.
follow ed by immersion in 0. 1 N sulphuric acid (H 2 S0 4 ) for 30, 45 , 60 and 90 min. At the end of each trcatmen t the sec::ds wc::re immed iately washed repeatedly in running water for 20 min to ensure comp lete remova l of any acid res id ue.

Surface sterilization
After the scarificati on treatment, seeds were disinfected with 2 g I-! Captan (15 m in) as fungic ide, followed by several treatments:  Treatment 3: Thc seeds were disinfected with hydrogen peroxide (H:P2) for 10 min followed by I or 2 g 1-1 mercuric chloride (HgCI 2 ) plus tween HO (5 ml) for 20 and 15 min, respec tively.

Statistical analysis
Results (recorded on difti.:rent days depending on the growth r~s pon scs be ing measured) were nna lysed by Duncan Multiple Range T I!st (DMRT) using the statistica l grapmc system of the Statistical GraphiCS Corporation computer programme and p < 0.05 constituted n signi ticallt d iffc r ent:~.

Results and Discussion
Preliminary results indicated that pre-soak ing of the seeds with Captan as fungicide was necessary. On the other hand, the use of Captan or Benlate at the same co ncentration (2 g I-I) and immersion time (15 min) showed no s ignifi cant differences between these fungicides ( p < 0.05) (dala not shown), although the effectiveness of Capt an and Benlate as fungicides in e liminating seed fungi is well recognized (Watts el al. 1993;George 1996). An early attempt to achieve in vitro and in silu seed germin at ion us in g imbi bed seeds wi th or w ithout testa a nd soaking intact seeds wi thout scarification did not sti mulate any germination . Scarification was effective with testa removal (Mederos el al. unpublished data). Moreover. hot water treatment for 15 min at 33°C, and 0.1 N sulphuric acid for 45 min, prior to disinfection, was effective for these Pistacia species and similar resu lts wit h hot water have been previousl y reported fro m other species (Ho i & van der Lind e 1992). After surface disinfecti on with treatment one, seeds of P. vera cv, male1fr, P. atlantica. P. lenliscus and P. lerebinlhus were still highly contaminated (8 3-100%) and browning exudates were considered and dismissed. In preliminary experi ments, ex hausti ve wash ing of the seeds in ascorbic acid so lution (0. 1 and 0.5 ~lM) for 30 and 45 m in, respectively after treatment one or two could not remove the contami nation and/or browning from the seeds. in direct contrast to the results obtained in a reca lcitrant Rh:inIls species (Mederos & Sc hobert 1995). The resu lts obtained in treatment lwo showed a significant effect on decontaminating seeds (F igure I). When seeds were treated with 1 g I-t HgC I 2 for 15 and 25 min between 42 and 83% Table 2 Effect of different cu lture medium containing 1 0 ~M AgN0 3 on percentage of germinated seeds and elongati on of seedlings. Dates are means and standard error (S .E.). When AgN0 3 was omitted from the media, germination was between 17 and 23% . All data were recorded at 25 and 43 days of culture  contamination was observed ( Figure lA). However, between 17% and 50% of seeds of these woody plants remained con taminated with 2 g I-I HgCI:z and 15 or 25 min immersion tim e. ivloreover, in the treatm ent s wi th I and 2 g I-I HgC I2 seeds of these Pistacia spec ies showed necros is (55% and 58%, respective ly).
The overa ll effect of disinfect ing the seeds with H 2 0 2 plus HgCI 2 depends on the concentrati on of the latter compound and th e immersion time as shown in experiment three ( Figure 2). Satisfacto ry decontaminati on percentages were obtained with treatment 3 (Fi gure 2A and B). The most sati sfactory resu lts were obtained in H,O, for 10 min plu s I g 1-' HgC l, for 20 min for P. vera cv. matellr and P. rerebinfhus ( Figure 2A). However, sterilising seeds with H 2 0, for 10 min plus 2 g 1-' HgCl, for 15 minutes proved best for P. atlantica and P. It:!lIfiscus ( Figure 28). We recom mended hydrogen peroxide treatment pri or to mercur ic c hloride application to mini mise tissue damage as a result of tile surface sterilization process. Moreover, du e to the toxic ity of HgCI 2 , Pistacia seeds mll st be rinsed th oroughly at least six times with sterile distill ed water prior to cultu re (Mederos 1991). Germination percentages were significantly lower when seeds were cu ltu red in th e norma l Heller, QL, MS culture media {between 17% and 23%} than w ith th e rest of the treatments. Under our preliminary experimental conditions the seeds excreted browning su bstan ces to the culture media and we observed very necroti c brown seeds after twenty fiv e days of culture. In these media , w ith AgNO), browning of the cu lture media was erad icated and the percentage of germination was stimulated. Simil ar results a re reported in organogenes is from juvenile shoot exp lants of Pistacia ar/ontica Desf. (Mede ros & Trujillo 199 8). As shown by the results presented in Table 2, the mo st sati sfactory results were obtained in the modified culture media supplemented with AgNO.l where the seeds did not excrete su bstances into any of th ese medi a. As far as the percentage of germinating seeds and elongation of the seedlings were concerned, growth on the modified Hell er medium (H + 7 mM KH 2 P0 4 ) was discernibly different if we compared the same parameters measured for seedlings 0 11 the modified QL an d MS med ia supplemented with 10 ~IM AgNO, (Table 2). AgNO, has a very slight pos itive effect on organogenesis and/or anti-browni ng eff~c t from different material plant (Housti eI al. 1992; George 1996). Best seedl ing growth was obtai ned 011 the modified Heller med ium for all the Pi,\'wcio species used (Table 2). Moreover, the elongatio n of the seedlings had stopped on day 43 of culture. The results of the comparison between these Pistacia species establi shed th at mod ified Hel ler sol uti on gave consiste ntl y better resu lts than any other macroelement solution using so lidified media. On the other hand , we found that a short pre-culture period usi ng normal or mod ifi ed Hell er (H + 7 111M KH}P0 4 + 10 11M AgN0 3 ) liquid medium for 15 to 17 days caused browning and necrosis of the Pis/acia seeds (83 -87%) and lower seed germination (7-13%) (Mederos el of. unpub lished data ).
The present resu lts indicate that in 1'ilro germi nation of seeds frol11 I'isillcia species is possible in the same cu ltu re mediulll but under different conditions. However, other investigations show that there are difference s in cxplant viability of PiSlacia species seedlings cultured on the same micropopagation medium (Gonzal ez & Frulos 1990: Mederos el al. I 994a,b, 1997aMederos & irujillo 1998). Different responses from di fferent lypes of explants (embryo, seed, apical and/or axillary shoot) may be due to the differeIH genotypes being cultured on the explant themselves. However, the results of the present study are encouraging, as the recalcitrant seeds of th ese Pis/acia species fail to germi nate ill situ and/or ;I11'ilrO after drying.