Plant Science, 70 (1990) 121 - 127 121 Elsevier Scientific Publishers Ireland Ltd.

CHARACTERIZATION OF PEA SHOOTS MINIATURIZED 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 s t ructures are least affected. Normal aUometric relationships are maintained. Miniaturization is achieved primarily through a reduction in relative ra te 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 par- ticularly striking example of this is the potato which, on an appropriate medium, produces tubers 3--5 mm 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 miniaturiza- tion 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 sati-

v u m L. When a single node is cultured on a nutrient medium containing cytokinin, an axil- lary 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). Inter- nodes 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 occa- sionally they contain a single, normal sized seed which is viable [5].

Shoot morphogenesis is believed to be gov- erned by a coordinated interplay of develop- mental 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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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) il lustrate 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 relat ive rate of elonga- tion (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 tha t of intact plants. (b) Growth centres in explants and intact plants contain comparable numbers of cells. However, the relative ra te 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.

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 morphol- ogy, and growth characteristics of axillary shoots in vitro with intact plants and axillary shoots in situ (on decapitated plants}.

Materials and Methods

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 indi- vidually in pots containing a bark-enriched pot- ting 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 -].

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 cham- ber.

Axillary shoots in vitro Surface-sterilised seeds were sown in auto-

claved vermiculite, moistened with sterile dis- tilled water, in 500 ml glass jars with white plastic screw-on lids. They were grown in a cul- ture 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 autoclav- ing. It was solidified with 5 g 1-1 agar. Each jar contained nodal explants from 5 shoots on 70 ml of medium.

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 trans- planted, 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 t reatment 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 third- oldest 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 num- bered 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 low- est 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 t reatment to anthesis were recorded.

A Uome try The lengths of structural components of the

third compound leaf and its subtending inter-

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

Growth rate The duration of growth and the daily rela-

tive 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 necessi- tated 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 inter- nodes 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 t reatment 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 petro- leum 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 signifi- cantly shorter (P < 0.001) in the axillary shoots (Table I). Mature lengths of internodes and

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Table I. Mean l eng ths (mm) ± 2 S.E. of m a t u r e o rgans on the t e rmina l shoot of in tac t peas, and s i tu and in vitro.

on ax i l la ry shoots grown in

In t ac t In s i tu In v i t ro

Shoot he ight I 135.3 ± 5.2 111.6 ± 5.1 58.8 + 2.5 Number of l eaves ' 11 7 13

In te rnode b 13.6 ± 0.8 13.4 ± 0.8 4.8 ± 0.4

St ipule c 24.1 ± 1.0 25.3 ± 1.4 7.0 ± 0.5 Pet io le ° 15.2 _+ 0.9 16.2 ± 1.1 5.9 ± 0.6 Prox imal leaf le t ~ 27.1 ± 1.3 23.4 ± 1.5 5.8 ± 0.7

Tendr i l c 38.6 ± 5.4 55.2 ± 4.0 11.0 ± 1.9 Node of 1st f lower d 10 6 10

W i n g p e t a l d 16.0 ± 0.4 14.8 _+ 0.4 11.2 _+ 0.5

• 28 days a f te r s t a r t of t r e a t m e n t . b Sub tend ing the th i rd o ldes t compound leaf.

c Borne on the th i rd o ldes t compound leaf. d Lowes t f lower 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 sub- stantially higher rate of elongation of the petiole on axillary shoots (Table II).

Because of these differences between ter- minal 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). Inter- nodes 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 h u > 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 develop- ment (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 signifi- cantly lower rates in vitro than in situ (Table II). Petioles ceased to elongate earlier in vitro than in situ h u < 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

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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 com- pound 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.

Intact In situ In vitro

Internode ~ RRE (days -1) 0.38 0.40 0.26 Duration (days) 18 19 19

Petiole a RRE (day -1) 0.34 0.54 0.24 Duration (days) 18 19 17

• Associated with the third-oldest compound leaf.

in those of in si tu shoots (•2; p < 0.001). Ep ide rma l cells in in te rnodes and pet ioles we re on a v e r a g e smal le r in v i t ro than the i r in situ coun t e rpa r t s by 38.5% and 23.5%, r e spec t ive ly (Table III). Cell n u m b e r was also reduced, by 42% in in te rnodes and, by 53% in pet ioles (Table III).

D i s c u s s i o n

Pea axi l lary shoots g rown in v i t ro a re not pe r fec t min ia tu res of the i r in si tu homologues. Al though all s t ruc tu ra l componen t s were reduced in size, d i f fe ren t o rgans were minia tur- ized to d i f fe ren t degrees . The size of reproduc- t ive s t r u c t u r e s such as the wing peta l (Table I ) , and the seed [5], is leas t a f fec ted by explant ing. This resu l t is cons i s ten t with the hypothes i s tha t the cu l ture med ium is deficient in one or more g rowth factors , a l though o ther possible explana t ions cannot be e l iminated. F e n n e r [11] sub jec ted intact shoots of Senecio vulgaris to va ry ing deg ree s of minera l nu t r i en t shor tage . The propor t ion of to ta l b iomass al located to reproduc t ion (seeds plus all anci l lary s t ruc- tures) was found to be v i r tua l ly unaffected, a l though the we igh t s of v e g e t a t i v e o rgans

decreased s ignif icant ly with decreas ing nu t r i en t supply.

The fo rmat ive rules which gove rn shoot morphogenes i s in situ are r e t a ined in cul ture. Mean al lometr ic cons tan t s for o rgans on shoots grown in v i t ro were comparab le to those of in situ shoots, which indicates t ha t p rocesses such as the par t i t ion ing of mer i s t ems , and posit ional informat ion as confer red by morphogene t i c gradients , are unaffected. Al lometr ic re la t ionships were s ignif icant ly more var iab le in v i t ro than in situ; exp lan t s seem to lack the fine-tuning tha t is so ev iden t in situ. This is p robab ly a consequence of the absence of roots , since explan ts lack the mach ine ry to se lec t ive ly control nu t r i en t uptake . McDaniel [12] has shown tha t root s y s t e m s d e t e r m i n e the ex t en t of g rowth of la tera l shoots in decap i ta ted tobacco plants .

Miniatur izat ion resu l t s p r imar i ly f rom a reduct ion in re la t ive r a t e of e longat ion (Fig. lb). When shoots are decapi ta ted , the axi l lary shoot which e m e r g e s exhibi ts c o m p e n s a t o r y g rowth [13]. This was par t icu lar ly pronounced in the petiole, which g rew 1.6 t imes more rapidly on axi l lary shoots in situ than on intact shoots (Table II). Clearly, when axi l lary buds are iso-

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

Intact In situ In vitro

Internode cell size 146 ± 4 148 ± 4 91 _+ 3 Cell number 93 90 53 Petiole cell size 235 ± 9 191 ± 6 146 _ 6 Cell number 65 85 40

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

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 minia- turization involves a reduction in the size of growth centres in the explants. When data from Tables I and II are substi tuted into the equation for exponential growth:

L = Lo e~t

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 t reatments 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 informa- tion, because they have been isolated from all other parts of the plant. Physical factors, such as osmotic potential, critical temperatures, and

127

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 G. Hussey and N.J. Stacey, In vitro propagation of potato (Solanum tuberosum L.). Ann. Bot., 48 (1981) 787-796.

2 E.F. George and P.D. Sherrington, Plant Propagation by Tissue Culture, Exegetics Ltd., Eversley, Basing- stoke, 1984, pp. 39-- 72.

3 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.

4 K.S. Gould, E.G. Cutter and J.P.W. Young, Morphoge- nesis of the compound leaf in three genotypes of the pea, Pisum sativum. Can. J. Bot., 64 (1986) 1268-- 1276.

5 G. Hussey and H.V. Gunn, Plant production in pea tPisum sativum L. cvs. Puget and Upton) from long- term callus superficial meristems, Plant Sci. Lett., 37 (1984) 143-148.

6 W. Halperin, Organogenesis at the shoot apex. Annu. Rev. Plant Physiol., 29 (1978) 239-262.

7 T. Murashige and F. Skoog, Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15 (1962) 473--497.

8 M. Ziv, A.H. Halevy and R. Shilo, Organs and plantlets regeneration of Gladiolus through tissue culture. Ann. Bot., (1970) 671 -- 676.

9 J.P. Stafstrom and I.M. Sussex, Pa t te rns of protein synthesis in dormant and growing vegetat ive buds of pea. Planta, (1988) 4 9 7 - 505.

10 J.H. Zar, Biostatistical Analysis, 2nd edn., Prentice- Hall, Inc., Englewood Cliffs, New Jersey, 1984, pp. 130 -131 .

11 M. Fenner, The allocation of minerals to seeds in Sene- cio vulgaris plants subjected to nutr ient shortage. J. Ecol., 74 (1986)385--392.

12 C.N. McDaniel, Influence of leaves and roots on meris- tem development in Nicotiana tabacum L. cv. Wiscon- sin 38. Planta, 48 (1980) 462 - 467.

13 W.P. Jacobs and B. Bullwinkel, Compensatory growth in Coleus shoots. Am. J. Bot. 40 (1953) 385 - 392.