Plant Cell, Tissue and Organ Culture 13:49-59 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

In vitro [14C]-labelled 6-benzyladenine uptake and 14C02 evolution in two Japanese plum cultivars

GRAZIA M A R I N O Istituto di Coltivazioni Arboree dell'Universit?t, 6 Via F. Re, 40126 Bologna, Italy

Received 5 May 1987; accepted in revised form 3 November 1987

Key words: Japanese plum, in vitro proliferation, 6-benzyladenine uptake, CO2 release

Abstract. Japanese plum cultivars 'Obilnaga' and 'Santa Rosa' showed different proliferation responses when grown in similar conditions in vitro. This led us to investigate BA uptake by shoots of both cultivars grown for different times (15 hrs, 1, 3, 6, 9, 13, 16 and 20 days) on an incubation medium enriched with 10 #Ci (370 kBq) 8-[t4C]BA/250ml. The decrease of radioactivity in the medium and its increase in the EtOH-soluble and -insoluble fractions of shoots of both cultivars grown for different times (15 h, 1, 3, 6, 9, 13, 16 and 20 days) on cultivars. Increasing amounts of ~4CO2 were detected in the culture atmosphere when shoots were grown in gas-tight vials. The type of container closure strongly affected proliferation. From the results reported here it is not possible to state whether different optimal subculture intervals and different proliferation responses of 'Santa Rosa' and 'Obilnaja' cultures are due to different tissue sensitivity to the cytokinin and/or to different metabolic activities. Neverthe- less the highest proliferation rates of both plums are clearly related to a drop of EtOH-soluble radioactivity of shoots by the end of subculture.

Introduction

From many studies on in vitro culture of fruit plant organs and tissues, it is evident that factors such as explant age, environmental conditions, agar brand and concentration, nutrients and especially hormone mixtures can influence shoot proliferation [5]. It is well-known that proliferation respon- ses to hormone types and concentrations vary widely among different fruit plant species and cultivars; apparently there is an opt imum hormonal con- centration for each plant on each medium [14]. However, all reports suggest that an exogenous cytokinin (alone or associated with an auxin) is generally required in the proliferation phase. Otherwise supraoptimal cytokinin con- centrations may cause abnormalities (e.g. vitrification) in many species [5, 7].

This paper is No. 620 of the Istituto di Coltivazioni Arboree. Part of the results were presented as a poster at the 15th International Symposium on Growth Regulators in Fruit Production, Rimini (Italy) 2 ~ September 1985. The research was partially supported by funds of the Ministry of Education, Roma (M.P.I. 60%).

50

As a consequence it seems logical to suppose that a better knowledge of the time course and rates ofcytokinin uptake could help in better understanding and thus improving of micropropagation.

Material and methods

Two Japanese plums, 'Santa Rosa' and 'Obilnaja', which have a similar high vigor of growth in the field, were cultured in vitro on Murashige & Skoog [11] revised salts medium, supplemented with (/~M): myo-inositol 555, ni- cotinic acid 4.06, pyridoxine-HC1 2.43, glycine 26.6, thiamine-HC1 2.96, 6-benzyladenine (BA) 3.11, indole-3-butyric acid 0.25, gibberellic acid 0.29, sucrose 58400 and (g l-L): agar (Bacto-Difco) 7; pH 5.7. The pH was adjusted with 0.1 N KOH before autoclaving at 120°C for 20 minutes (medium A). Subcultures occurred every four weeks.

Single shoots, about 15 mm long, derived from 2 to 3-year-old cultures of both cultivars with steady proliferation rates, were used in the following trials.

Trial 1. To determine the shortest subculture interval (the shorter the production time the better for commercial purposes), shoots were grown for different times (15 hours, 1, 3, 6, 9, 13, 16 and 20 days) on medium A. They were then removed from medium A, washed for a few minutes with sterile water to remove free BA [15] (i.e. agar fragments sticking at the shoot base) and then transferred to fresh medium with the same composition except t h a t BA was omitted (medium B). Transfer to medium B allowed new shoots, induced on medium A, to grow enough to be dissected and counted.

Fresh and dry weights were measured at each sampling time (15 hours, 1, 3, 6, 9, 13, 16 and 20 days). The proliferation responses were determined after a 20-day total interval of subculture (period on medium A + that on medium B) on 15 shoots per treatment, 5 per each 500 ml jar. The experi- ment was repeated twice; data on proliferation reported below are the averages of the two repetitions.

Trial 2. To investigate BA uptake, shoots were incubated on the same medium as medium A supplemented after autoclaving with 10/~Ci (370 kBq) 8-[~4C]BA (Amersham)/250ml, filter(0.22 /~m)-sterilized (medium A-L4C contains: BA 3.11 pM + 14C-BA). Samples of 12 shoots (6 shoots per 50ml plastic vessel, diameter 53mm, each containing 10ml of medium) were collected at different times (15 hours, 1, 3, 6, 9, 13, 16 and 20 days), washed,

51

blotted, weighed and stored at - 20°C until being extracted in 70% ethanol (by grinding in a mortar, leaving the homogenate at 4°C for 12 hours, then centrifuging at 6000 rpm for 10 minutes). The supernatant was recovered and the precipitate was reextracted in ethanol in the same way. The two supernatants were combined and evaporated to the aqueous phase under vacuum [6] at room temperature (about 18°C). For liquid scintillation counting, 1 ml of each sample-supernatant was added to 10 ml of aqueous counting scintillant (Amersham) and counted in a 5 Searle liquid scintil- lation counter. No purification of the aqueous extracts was required because their quench was very low. The experiment was repeated three times; data reported on EtOH-soluble shoot radioactivity represent the averages of the three repetitions.

In the last replicate, EtOH-soluble radioactivity of the medium after shoot removal was also determined by freezing at - 20°C, stirring with 70% ethanol, leaving at 4°C for 12 hours, filtering on Whatman No. 1 paper, evaporating to the aqueous phase and subjecting the filtrates to the same counting procedures described above. Data were not statistically analysed as referred to a single value for each treatment (media from two vessels were mixed and extracted together at each time).

Trial 3. C O 2 release and the incorporation of 14C in the EtOH-insoluble fractions of medium and tissue were also measured. Shoots were cultured singly in 40 ml vials that were sealed with gas-tight neoprene plugs, each vial containing 1.5 ml of medium A-14C.

Seven vials were chosen at random at each of five different times (3, 6, 13, 16 and 21 days). Before opening the vials, samples of the culture atmosphere were taken in 10 ml syringes, each containing 5 ml of Carbosorb able to trap 14CO2. Before counting in the scintillation counter, 7.5ml of Permafluor were added to each 5 ml Carbosorb volume.

Later shoots and media were extracted individually as described above. The radioactivity in the EtOH-insoluble fractions was determined by drying the shoot pellets and the residue of the medium and burning them in a Packard Tri-Carb sample oxidizer. Data reported on radioactivity in the shoots and medium and ~4CO2 evolution represent the averages of the seven replications.

At each time, 14CO2 release and radioactivity of medium not inoculated with shoots were measured also.

Micropropagation techniques and growth room conditions were the same as previously reported for 'Santa Rosa' [9].

52

Results and discussion

Shoot growth

A large increase in proliferation occurred in both plums grown for 3 days

on medium A compared to those that remained in contact with BA for only 1 day (Fig. 1). At least 13 days were required by 'Santa Rosa ' shoots to reach the highest proliferat ion rate (Duncan 's test P = 0.05: the proliferation rate at 20 days - 7.0 + 0.6 - was the only one higher than that at 9 days -

5.1 ___ 0.5 - but was not different f rom that at 13 days - 0.65 ___ 6). 'Obil- naja' had a much higher proliferation rate than 'Santa Rosa' , that cont inued to increase until the end of subculture (25.2 ___ 1.6). Both 'Santa Rosa ' and 'Obilnaja ' shoots that remained on medium A for less than 3 to 6 days became chlorotic. Since kinetin t reatment is known to delay senescence and chlorophyll degradat ion in detached leaves [13], this response indicates the

necessity o f maintaining these cultures on a medium containing cytokinin. Moreover , at least 13 days were generally needed to obtain most of re- generated shoots large enough to be dissected before transfer to medium B.

m

c 3 0

® c 0

• , , 2 5 ,

"0 0 0

2 0 . L. Q.

I i 1 0 0

o .

b

• O b i l n a j a

0 S a n t a R o s a

0 1 3 6 9 13 16 2 0

t ime on medium wi th BAP ( d a y s )

Fig. 1. Shoot proliferation (as axillary buds) after different intervals on BA 3.11 pM (medium A) and transfer to a medium lacking BA (medium B). Proliferation checked after a total subculture interval of 20 days (period on medium A plus that on medium B). Data are average of two replications. Bars represent SEM.

53

Increase in fresh weight over time was quite large in shoots of both plums, being higher for 'Obilnaja' at most of the points considered (Fig. 2). Dry weight of 'Obilnaja' shoots increased from 7% (initial d wt) to 11% by day 13, and decreased to about 9.5% after that time. 'Santa Rosa' shoots had a higher dry weight, which was about 16% after 9 days, and later gradually decreased to values similar to those of 'Obilnaja' tissue by the end of subculture.

When shoots were cultured in air-tight vials, as observed by others [2, 3] this kind of closure was responsible for a reduction of shoot proliferation and growth. Shoot production after 21 days of culture on BA-enriched medium was 2.3 + 0.4 and 3.4 _+0.7 in 'Santa Rosa' and 'Obilnaja' respec- tively. The new shoots grew enough to be countable only by the end of subculture.

BA uptake

Plant radioactivity is expressed as dpm/shoot because the large increase in fresh and dry weight and the fact that it was different in 'Obilnaja' and 'Santa Rosa' shoots (due to different moments when shoot proliferation and leaf expansion occurred) would make the data expressed on a weight basis unclear. Similar data were presented on a per shoot basis by others for elm and apple shoots [1, 12].

EtOH-soluble radioactivity of shoots grown in vessels (Fig. 3) increased quickly in both plums until 9 days after transfer to the incubation medium

5

J

m | ,

m w . 3

. 2

0 Santa Rosa • Obiinaja

t i m e on med ium with B A ( d a y s )

Fig. 2. Increase in fresh weight of'Santa Rosa' and 'Obilnaja' shoots grown on medium A (BA 3.11 #M) for different intervals. Data are average of three replications. Bars represent SEM.

54

4.0

3.5

3.0

~ 2.5

~ 2.0

.~.

~ 1.5

_3 1.o,

z 0

"'o.s,

i

O S&nt& Rosa • Obllna|a

• i 3 6 9 13 1~, 2"0 t i m e on m e d i u m wi th 14C- BA ( days )

Fig. 3. Tissue EtOH-soluble radioactivity after growing shoots in vessels on the incubation medium (medium A, plus 10/zCi 8-[~+C]BA/250 ml) for different intervals. Data are average of three replications. Bars represent SEM.

and dropped after 13 and 16 days in 'Obilnaja' and 'Santa Rosa' respective- ly. Differences between the two cultivars were not significant at any time. In Fig. 4 the EtOH-soluble radioactivity of the medium and shoots is reported, but only from the third replicate of this experiment. Radioactivity of the medium is expressed as available radioactivity (dpm/shoot: the ratio of the total radioactivity of the medium and the number of shoots grown on it) in order to compare the decrease of 8-[~4C] BA in the medium with the actual radioactivity detectable in the shoots at each time. Owing to the small volume of medium contained in each vessel, no concentration gradient around shoots due to the agar gel [16] was considered, and BA was thus assumed to be equally distributed in the medium. Radioactivity of the medium was about the same for the two plums up to one day after shoot transfer to the medium m-]4C; thereafter it decreased in both plum cultures, apparently being always lower in 'Obilnaja'.

Radioactivity in the EtOH-soluble and EtOH-insoluble fractions of shoots grown in gas-tight vials increased in the two plum cultivars during the

55

3 . 0

o

"~ 2 .5

E 0. 2 . 0 .

0 1.5,

~ 1.0

, Z ~ 0.5

'~ 0

0 Santa Rosa • Obilnaja

?- . . . . . . . . . . . . . . . . . . . o

6 "i ~ e ~ ~3 l"a 2"0 t ime on medium with 14C - B A ( d a y s )

,11

,10~

E ,9

e . . c :

7 ' g : ^ o

5 +g+.

o + +

3 _1

1 1

Fig. 4. Medium and tissue EtOH-soluble radioactivity after growing shoots in vessels on the incubation medium (medium A, plus I0 #Ci 8-[~4C]BA/250ml) for different intervals. One replication only.

whole culture time, being higher in 'Santa Rosa' at several points (Fig. 5). Radioactivity in the EtOH-soluble fraction of the medium significantly decreased during the proliferation phase (Fig. 6), thus confirming the results of the previous trial (Fig. 4); no difference was observed between the values for 'Santa Rosa' and 'Obilnaja'. In contrast the radioactivity in the insoluble fraction remained constant and tended to be higher in 'Obilnaja' on the last three sampling dates (Fig. 6).

In medium not inoculated with shoots, radioactivity did not change during 21 days in the growing chamber, EtOH-insoluble radioactivity being about 10% of the total; that was probably due to a constant contamination by the ethanolic extract.

C02 release

It was supposed for elm shoots that a considerable amount of 14CO2 should be released during the in vitro culture through purine degradation to allan- toin, allantoic acid and urea [1]. Otherwise, attempts to trap CO2 in in vitro apple shoot cultures growing in an air-circulating apparatus were unsuccess-

56

ZZ6

u l w

o

0 Santa ~oaa • ObllnaJa

~T~ " ' ° ° " * ' " ' ° ° " * ° ~

. . . . . . . . = : : : : : : : : * " : .o-"

3 6time on 9 16 21 medium with 1314C-BA ( days )

Fig. 5. Shoot radioactivity after growing cultures in air-tight vials on the incubation medium (medium A, plus 10 #Ci 8-[~4C]BA/250ml) for different intervals. Data are average of seven replications. Bars represent SEM.

Z Z ,6 o o ww

- - 5

=,4,

m

..~ "o

i . •

_o E

0 Santa Rosa • Obilnarja

6 9 13 16 2"1 t ime on medium wi th 14C-BA ( days )

Fig. 6. Medium radioactivity after growing cultures in air-tight vials on the incubation medium (medium A, plus 10 #Ci 8-[~4C]BA/250 ml) for different intervals. Data are average of seven replications. Bars represent SEM.

57

ful [ 12]. By the method described above it was possible to detect increasing amounts of ~4CO2 in the culture atmosphere of cultures of both plum cultivars (Fig. 7). Data are given on a per shoot basis so as to be able to compare the increase in shoot radioactivity and the decrease in medium radioactivity to the loss of ~4C as ~4CO 2 when shoots were cultured singly. From day 3 to day 13 the amount of ~4CO2 measured increased gradually and steadily; then it doubled by day 16 and later it resumed the original gradual rate of increase until day 21.

No 14C02 was detected in the control vials (no shoots). The decrease of radioactivity in the EtOH-soluble fraction of the medium

(Figs. 4 and 6) and the increase of radioactivity in the shoots (Figs. 3, 4 and 5), at least in the first part of the proliferation phase (Figs. 3 and 4), reveal a rapid and considerable BA uptake.

Applied cytokinins are known to undergo a very rapid metabolism in different plants and tissues [8, 10, 16]. In the shoots cultured in standard conditions a similar metabolism could be hypothesized because of the decrease of their EtOH-soluble radioactivity by the end of subculture (Fig. 3), which corresponded to the increase in proliferation (Fig. 1). This hypo- thesis was supported, in the experiment where gas-tight vials were used, by the evolution of t4 CO2 (Fig. 7) and the increase of radioactivity in the EtOH- insoluble fraction of the shoot extracts (Fig. 5), the latter probably due to

1.5

o ~1,0

i "u ;

~ o . 5 .

o Sa.ta Rosa l

6 9 1"3 14 C 16 2"1 t i m e o n m e d i u m w i t h - B A ( days )

Fig. L ['4C]-labelled carbon dioxide evolution in the culture atmosphere over different shoot culture times. Data are average of seven replications. Bars represent SEM.

58

BA incorporation into compounds, e.g. nucleic acids [4], not extractable by the method used. The reason that in these stress conditions no decrease in radioactivity in the shoots was measured over time, as in Fig 3, might be that BA metabolism was reduced. In fact, the growth of both plums was very low; 'Obilnaja' was especially affected with a reduction in proliferation rate from about 25:1 to 3:1. Otherwise it is also possible that the carbon was reabsorbed as CO2 into the plants.

In conclusion, proliferation rates of 'Santa Rosa' and 'Obilnaja' are examples of the range of responses to cytokinin in in vitro cultured shoots. However, from these results it is not possible to state whether different optimal subculture intervals (13 days for 'Santa Rosa' and 20 days for 'Obilnaja') and diverse proliferation rates are due to varying tissue sensitiv- ity to the cytokinin and/or to different metabolic activities. Nevertheless the highest proliferation rates of both plums are related to a drop of EtOH- soluble radioactivity of shoots at the end of subculture. Moreover, when a similar decrease did not occur, as in gas-tight vials, proliferation always remained very low.

It is thus clear that investigations on uptake and metabolism of hormonal substances can help to enhance understanding of culture growth and the requirements needed to improve proliferation.

Acknowledgements

The author thanks Dr. E. Magnanini for the kind help in using the sample oxidizer and Miss Ancherani M. for the technical cooperation.

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