Characterization of pea shoots miniaturized by growth in culture

Plant Science, 70 (1990) 121 - 127

121

Elsevier Scientific Publishers Ireland Ltd.

C H A R A C T E R I Z A T I O N OF P E A SHOOTS M I N I A T U R I Z E D BY GROWTH IN CULTURE

KEVIN S. GOULD and STEPHEN R.P. CHAMBERS

Department of Botany, University of Auckland, Private Bag, Auckland fNew ZealandJ (Received December 18th, 1989) (Revision received April 4th, 1990) (Accepted April 10th, 1990)

When nodal segments of pea (P/sum sativum L.) are cultured in vitro, axillary shoots grow into miniature versions of their in situ homologues. Different organs on these shoots are miniaturized to different degrees. Leaf tendrils are most reduced in size; reproductive structures are least affected. Normal aUometric relationships are maintained. Miniaturization is achieved primarily through a reduction in relative rate of elongation. Both cell size and cell numbers are reduced in the epidermis.

Key words: miniaturization; in vitro shoots; growth rates; allometry; morphogenesis; Pisum sativum

Introduction Shoots cultured in vitro very often grow into miniature versions of the parent plant. A particularly striking example of this is the potato which, on an appropriate medium, produces tubers 3--5 m m in diameter borne on small upright stolons [1].The miniaturization process is at first beneficial to the micropropagator because only a limited area is required to maintain or multiply large numbers of plantlets [2]. Ultimately, however, the small stature of explants is disadvantageous because a long growth period is required to provide plants of a suitable size for retail [2].The causes and the mechanism of the process by which miniaturization is achieved have never before been reported. In this paper we examine the miniaturization process of lateral shoots in the pea, P i s u m sativ u m L. When a single node is cultured on a nutrient medium containing cytokinin, an axillary shoot is induced to grow out. The actual degree to which this shoot elongates depends upon its original position on the parent shoot, as well as the level of cytokinin available [3]. However, for any set of culture conditions, the explant will always grow to a greater or lesser

extent into a condensed version of the parent plant (with the notable absence of roots). Internodes and leaves are reduced in both size and number. Changes in the complexity of leaves along the in vitro shoot recapitulate, albeit in a more rapid succession, the heteroblastic series of intact plants [4]. Shoot cultures eventually form small flowers which give rise to pods [3,5]. Pods are normally empty and abort, but occasionally they contain a single, normal sized seed which is viable [5]. Shoot morphogenesis is believed to be governed by a coordinated interplay of developmental signals, the synthesis and distribution of which are tightly regulated over both time and space [6]. The close homology between nodal explants and intact pea shoots would suggest that nascent primordia in the explant receive, and are competent to respond to all of the normal developmental signals. There are at least three possible pathways which lead to miniaturization (Fig. 1): (i) organ primordia have smaller growth centres in explants than in intact plants (Fig. la); (ii) explants and intact plants have comparable growth centres, but in vitro, these grow at a much reduced rate (Fig. lb); (iii) explants and intact plants have comparable growth centres which grow at simi-

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122

lar rates, but which, in vitro, grow for a shorter duration (Fig. lc). Of course, any combination of the above is possible. We have examined these possibilities by comparing the allometry, mature morphology, and growth characteristics of axillary shoots in vitro with intact plants and axillary shoots in situ (on decapitated plants}.

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Intact plants Seeds of the dwarf pea, P. sativum L. cv. W.F. Massey, were obtained from Yates New Zealand Ltd. (Auckland}. They were sown individually in pots containing a bark-enriched potting compost. The emergent shoots were grown in a constant environment chamber at 23 _+ 1 °C, and an 18-h photoperiod was provided by cool white fluorescent lamps with a photon fluence rate of 50 ~mol m -2 s -].

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Materials and Methods

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Axillary shoots in situ Intact plants grown for 10 -- 14 days as above were decapitated above node 3. All buds borne in the leaf axil at node 3 were removed. One lateral shoot emerged from the leaf axil at node 2 in each decapitated plant. These axillary shoots were grown adjacent to the intact plant population in the constant environment chamber.

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TIIE Fig. 1. Possible routes leading to miniaturization of internodes and petioles on pea shoots growing in vitro. The model assumes that a discrete, steady-state growth centre is intercalated between two non-growing regions. Curves in (a) and (b) illustrate spatial distributions of growth for any one point in time. In (c), temporal changes in the growth profile are shown for the organ as a whole. Solid line, intact plants; broken line, explants. (a) The relative rate of elongation (R.R.E.) of a cell in the growth centre is similar for explants and intact plants. However, because fewer cells contribute to the growth of explants, overall growth is less than t h a t of intact plants. (b) Growth centres in explants and intact plants contain comparable numbers of cells. However, the relative rate of elongation of these cells is lower in vitro than in situ. (c) The growth centres in explants and intact shoots contain comparable numbers of cells, and they grow at comparable rates. However, the duration of growth is shorter in the explants.

Axillary shoots in vitro Surface-sterilised seeds were sown in autoclaved vermiculite, moistened with sterile distilled water, in 500 ml glass jars with white plastic screw-on lids. They were grown in a culture room for 10 days under temperature and light regimes comparable to the intact and decapitated plant treatments. Nodal explants were taken exclusively from node 2. They were cultured in fresh jars on Murashige and Skoog's medium [7] as modified by Ziv et al. [8] and Hussey and Gunn [5]. The medium contained 6-benzylaminopurine at 0.75 mg 1-1, which was introduced prior to autoclaving. It was solidified with 5 g 1-1 agar. Each jar contained nodal explants from 5 shoots on 70 ml of medium.

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Decapitated plants in vitro A further population of the plants grown for 10 days in vermiculite under aseptic conditions were decapitated above node 3 and then transplanted, with seed and roots intact, onto the nutrient medium described above. After 28 days in culture, these plants had produced many small shoots, resulting from the outgrowth of lateral buds. It was not possible to identify a leader shoot in these cultures. This treatment did not, therefore, serve as a useful control for examining the influence of the seed and roots on plant stature, and the plantlets were not examined further. Mature morphology The morphology of 25 shoots from each treatment was examined 28 days after sowing (intact plants), decapitating (in situ shoots), or explanting in vitro. Parameters measured included: shoot height, total leaf number, and lengths of structural components of the thirdoldest compound leaf and of its subtending internode. This leaf was chosen because it was present as the youngest leaf primordium on the embryonic axis in the dormant seed, and on the largest axillary bud prior to shoot decapitation or nodal explanting. This leaf had, therefore, grown almost entirely under the conditions imposed by each treatment. Nodes were numbered acropetally above the seed cotyledons (intact plants), basal callus (in vitro shoots), and from the point of insertion of the axillary shoot in decapitated plants. In intact plants the third compound leaf occurred at node 5 (the two lowest leaves were scalar, all higher leaves were compound). For axillary shoots both in situ and in vitro the third compound leaf occurred most commonly at node 3, but occasionally at node 4. Further populations of shoots were grown until the first flower had fully opened. The length of the wing petal and the duration from the start of the treatment to anthesis were recorded. A Uome try The lengths of structural components of the third compound leaf and its subtending inter-

node were measured in a further 100 shoots from each treatment. Measurements encompassed the full course of development, from late primordial stages to maturity.

Growth rate The duration of growth and the daily relative rate of elongation of the internode and petiole associated with the third compound leaf were recorded for 25 shoots of each treatment. Measurements of the youngest stages necessitated bending back the outer stipules. Shoots grown in vitro were measured carefully in a laminar flow chamber, and were returned to their original position on the nutrient medium after each measurement. Neither the bending of stipules, nor the removal of shoots from the culture jar affected the final length of internodes and leaves. Measurements were taken daily using a ruler. Growth rates were determined over 5 days during the exponential phase. Cell lengths The length of epidermal cells in the central portion of both the mature internode and petiole were determined from nail varnish replicas using a microscope and drawing tube. Ten shoots of each treatment were used, 50 cells were measured in each. Results

There are three buds in the axil of the leaf at node 2 of intact pea shoots. It is the largest of these which grows out preferentially upon decapitation or explanting in vitro [9]. This was confirmed in the current study by marking buds in a number of plants with carbon in petroleum jelly immediately prior to the treatment. Axillary shoots grown in situ under the experimental conditions described were not morphologically identical to the terminal shoot of intact plants. Fewer leaves were produced, leaf tendrils were significantly longer h° < 0.001), and leaflets and flowers were significantly shorter (P < 0.001) in the axillary shoots (Table I). Mature lengths of internodes and

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Table I.

Mean l e n g t h s (mm) ± 2 S.E. of m a t u r e o r g a n s on t he t e r m i n a l shoot of i n t a c t peas, and on a x i l l a r y s hoot s g r o w n in s i t u and in vitro. Intact Shoot h e i g h t I N u m b e r of l e a v e s ' Internode b Stipulec Petiole° P r o x i m a l leaflet ~ Tendrilc Node of 1st flo wer d Wingpetald

135.3 11 13.6 24.1 15.2 27.1 38.6 10 16.0

± 5.2 ± 0.8 ± 1.0 _+ 0.9 ± 1.3 ± 5.4 ± 0.4

In s i t u

In v i t r o

111.6 7 13.4 25.3 16.2 23.4 55.2 6 14.8

58.8 13 4.8 7.0 5.9 5.8 11.0 10 11.2

± 5.1 ± ± ± ± ±

0.8 1.4 1.1 1.5 4.0

_+ 0.4

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0.4 0.5 0.6 0.7 1.9

_+ 0.5

• 28 d a y s a f t e r s t a r t of t r e a t m e n t . b S u b t e n d i n g t h e t h i r d o l d e s t compound leaf. c Borne on t h e t h i r d o l d e s t compound leaf. d L o w e s t flo wer to open.

petioles were not affected, but the allometric ratio of lengths of petiole to internode was significantly greater (P ~ 0.02) for the axillary shoots during the entire course of development (Figs. 2a and 2b). This was the result of a substantially higher rate of elongation of the petiole on axillary shoots (Table II). Because of these differences between terminal and axillary shoots, only the growth of the latter can be regarded as the developmental potential of a nodal segment cultured in vitro. Axillary shoots on nodal explants elongated for 21--28 days in culture. They produced more leaves than the in situ shoots at a similar age, and flowered at a later date and at a higher node (Table I). Every structural component of the explant was reduced in size relative to the axillary shoots in situ (Table I). However, different organs were reduced to different degrees. The flower was least reduced in size (24%), and the leaf tendril was the most reduced (800/0). Internodes and petioles were each reduced by 640/0. Ratios of lengths of petiole to internode over the full course of development (Figs. 2b and 2c) were significantly more variable in vitro than in situ (F-test of linear regression, P ~ 0.001). Taking the heterogeneity of variances into account by using Welch's approximate t value [10], the regression coefficients (i.e., allometric

constants) for in vitro and in situ shoots were not statistically different hu > 0.05). Allometric plots of length of the proximal leaflet over length of the rachis (i.e., petiole plus tendril) provide an index of the evolving shape of the leaf over the course of development (Figs. 3a-- 3c). For leaves borne on the terminal shoot of intact plants and on axillary shoots in situ, these plots were biphasic, the allometric constant being significantly higher at rachis lengths below 15 mm (Figs. 3a and 3b). Leaf shape was significantly more variable in explants, and there was no evidence of a biphasic relationship (Fig. 3c). The regression coefficient for leaves on in vitro shoots did not differ significantly from the lower part of the regression for leaves on in situ shoots (Welch's approximate t; P > 0.1). Internodes and petioles elongated at significantly lower rates in vitro than in situ (Table II). Petioles ceased to elongate earlier in vitro than in situ hu < 0.05). However, the reduction in duration of elongation was small (10.5%) in comparison to the reduction in relative rate of elongation (56%). The duration of elongation of internodes was not affected by explanting. Epidermal cell lengths were not normally distributed within each population sampled. There were significantly more smaller cells in internodes and petioles of in vitro shoots than

125

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LENGTH OF IN'rEI:INOOE (m) Fig. 2. Allometric relationship between the lengths of pea internodes and petioles in (a) intact plants, (b) axillary shoots in situ, (c) axillary shoots in vitro, k, allometric constant.

Fig. 3. Allometric relationship between the lengths of the proximal leaflet and the rachis of the third-oldest compound pea leaf in (a) intact plants, (b) axillary shoots in situ, (c) axillary shoots in vitro, k, allometric constant.

126 Table II. Mean relative rate of elongation (RRE) and mode duration of elongation for pea internodes and petioles in terminal shoots of intact plants, and in axillary shoots grown in situ and in vitro.

Internode ~ Petiole a

RRE (days-1) Duration (days) RRE (day-1) Duration (days)

Intact

In situ

In vitro

0.38 18 0.34 18

0.40 19 0.54 19

0.26 19 0.24 17

•Associated with the third-oldest compound leaf.

in t h o s e of in situ shoots (•2; p < 0.001). E p i d e r m a l cells in i n t e r n o d e s and petioles w e r e on a v e r a g e s m a l l e r in v i t r o t h a n t h e i r in situ c o u n t e r p a r t s b y 38.5% and 23.5%, r e s p e c t i v e l y (Table III). Cell n u m b e r was also reduced, by 42% in i n t e r n o d e s and, by 53% in petioles (Table III). Discussion

P e a axillary shoots g r o w n in v i t r o a r e not p e r f e c t m i n i a t u r e s of t h e i r in situ homologues. A l t h o u g h all s t r u c t u r a l c o m p o n e n t s w e r e r e d u c e d in size, d i f f e r e n t o r g a n s w e r e miniaturized to d i f f e r e n t d e g r e e s . T h e size of r e p r o d u c tive s t r u c t u r e s such as the wing p e t a l (Table I), and the seed [5], is l e a s t affected by explanting. This r e s u l t is c o n s i s t e n t with t h e h y p o t h e s i s t h a t the c u l t u r e m e d i u m is deficient in one or m o r e g r o w t h factors, a l t h o u g h o t h e r possible e x p l a n a t i o n s cannot be eliminated. F e n n e r [11] s u b j e c t e d intact shoots of Senecio vulgaris to v a r y i n g d e g r e e s of m i n e r a l n u t r i e n t s h o r t a g e . The p r o p o r t i o n of t o t a l b i o m a s s allocated to r e p r o d u c t i o n (seeds plus all ancillary structures) was found to be v i r t u a l l y unaffected, although the w e i g h t s of v e g e t a t i v e o r g a n s

decreased significantly with decreasing n u t r i e n t supply. T h e f o r m a t i v e rules which g o v e r n shoot m o r p h o g e n e s i s in situ are r e t a i n e d in culture. Mean allometric c o n s t a n t s for o r g a n s on shoots g r o w n in v i t r o w e r e c o m p a r a b l e to those of in situ shoots, which indicates t h a t p r o c e s s e s such as the p a r t i t i o n i n g of m e r i s t e m s , and positional information as c o n f e r r e d by m o r p h o g e n e t i c gradients, are unaffected. Allometric relationships w e r e significantly m o r e v a r i a b l e in v i t r o t h a n in situ; e x p l a n t s s e e m to lack the fine-tuning t h a t is so e v i d e n t in situ. This is p r o b a b l y a consequence of the a b s e n c e of roots, since e x p l a n t s lack the m a c h i n e r y to s e l e c t i v e l y control n u t r i e n t u p t a k e . McDaniel [12] has shown t h a t root s y s t e m s d e t e r m i n e the e x t e n t of g r o w t h of lateral shoots in d e c a p i t a t e d tobacco plants. Miniaturization r e s u l t s p r i m a r i l y f r o m a reduction in r e l a t i v e r a t e of elongation (Fig. lb). When shoots are d e c a p i t a t e d , the axillary shoot which e m e r g e s exhibits c o m p e n s a t o r y g r o w t h [13]. This was p a r t i c u l a r l y p r o n o u n c e d in the petiole, which g r e w 1.6 t i m e s m o r e rapidly on axillary shoots in situ t h a n on intact shoots (Table II). Clearly, w h e n axillary buds are iso-

Table III. Epidermal cell length (~m) ± 2 S.E. and cell numbera in mature internodes and petioles on the terminal shoot of intact plants, and on axillary shoots grown in situ and in vitro.

Internode cell size Cell number Petiole cell size Cell number

Intact

In situ

In vitro

146 ± 4 93 235 ± 9 65

148 ± 4 90 191 ± 6 85

91 _+ 3 53 146 _ 6 40

aValue obtained when the mature length (Table I) is divided by the cell length.

127

lated and cultured on a nutrient medium, they lose this extra growth potential. The duration of elongation of petioles was also slightly reduced under the in vitro conditions. This may not be important, because internodes achieved a comparable degree of miniaturization without any reduction in the duration of growth. There is no evidence to suggest that miniaturization involves a reduction in the size of growth centres in the explants. When data from Tables I and II are substituted into the equation for exponential growth:

the gaseous environment may also play a role. Work is in progress to elucidate the mechanism involved. Acknowledgements This work was supported in part by two University of Auckland Research Committee grants, #0408.046 and #0408.051. References 1

L

= Lo e~t 2

where, L m = mature length of organ, L ° = original length immediately prior to exponential growth, r = relative rate of elongation and t = duration of elongation; the original lengths (L o) of internodes and petioles prior to exponential growth are found to be actually larger in vitro than in situ. Such calculations are only approximations, because they do not account for the period of deceleration in growth rate which succeeds the exponential growth phase. (Our data indicated that there was no difference among treatments in the duration of the deceleration phase. The rate of deceleration may have been affected, however). They do indicate that explants have an adequate 'biological capital' of cells at the onset of the exponential growth phase, but the growth potential is not realised because of the treatments imposed. Both cell division and cell elongation in the epidermis are adversely affected by the treatment. It is unlikely that explants are miniaturized solely because of deficiencies in the nutrient medium. The phenomenon of miniaturization is widespread among plant species, even though many different nutrient media have been employed. Explants lack correlative information, because they have been isolated from all other parts of the plant. Physical factors, such as osmotic potential, critical temperatures, and

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4

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G. Hussey and N.J. Stacey, In vitro propagation of potato (Solanum tuberosum L.). Ann. Bot., 48 (1981) 787-796. E.F. George and P.D. Sherrington, Plant Propagation by Tissue Culture, Exegetics Ltd., Eversley, Basingstoke, 1984, pp. 39-- 72. K.S. Gould, E.G. Cutter, J.P.W. Young and W.A. Charlton, Positional differences in size, morphology, and in vitro performance of pea axillary buds. Can. J. Bot.,65 (1987)406--411. K.S. Gould, E.G. Cutter and J.P.W. Young, Morphogenesis of the compound leaf in three genotypes of the pea, Pisum sativum. Can. J. Bot., 64 (1986) 1268-1276. G. Hussey and H.V. Gunn, Plant production in pea tPisum sativum L. cvs. Puget and Upton) from longterm callus superficial meristems, Plant Sci. Lett., 37 (1984) 143-148. W. Halperin, Organogenesis at the shoot apex. Annu. Rev. Plant Physiol., 29 (1978) 2 3 9 - 2 6 2 . T. Murashige and F. Skoog, Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15 (1962) 473--497. M. Ziv, A.H. Halevy and R. Shilo, Organs and plantlets regeneration of Gladiolus through tissue culture. Ann. Bot., (1970) 671 -- 676. J.P. Stafstrom and I.M. Sussex, P a t t e r n s of protein synthesis in dormant and growing vegetative buds of pea. Planta, (1988) 4 9 7 - 505. J.H. Zar, Biostatistical Analysis, 2nd edn., PrenticeHall, Inc., Englewood Cliffs, New Jersey, 1984, pp. 130 -131. M. Fenner, The allocation of minerals to seeds in Senecio vulgaris plants subjected to nutrient shortage. J. Ecol., 74 (1986)385--392. C.N. McDaniel, Influence of leaves and roots on meristem development in Nicotiana tabacum L. cv. Wisconsin 38. Planta, 48 (1980) 462 - 467. W.P. Jacobs and B. Bullwinkel, Compensatory growth in Coleus shoots. Am. J. Bot. 40 (1953) 385 - 392.